Peptide conjugated particles

ABSTRACT

The present invention provides compositions comprising peptide-coupled biodegradable poly(lactide-co-glycolide) (PLG) particles. In particular, PLG particles are surface-functionalized to allow for coupling of peptide molecules to the surface of the particles (e.g., for use in eliciting induction of immunological tolerance).

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 16/847,450,filed Apr. 13, 2020 which is continuation of application Ser. No.15/922,241, filed Mar. 15, 2018 which is a continuation of applicationSer. No. 15/341,935, filed Nov. 2, 20216 which claims priority to U.S.Provisional Patent Application No. 61/865,389, filed Aug. 13, 2013, U.S.Provisional Patent Application No. 61/869,279, filed Aug. 23, 2013 andU.S. Provisional Patent Application No. 61/887,112, filed Oct. 4, 2013.This application is also related to PCT Application No.PCT/US2013/047079 filed Jun. 21, 2013 which claims priority to U.S.Provisional Application No. 61/662,687, filed Jun. 21, 2012. Thecontents of each of which is hereby incorporated by reference in itsentirety.

FEDERAL SUPPORT

This invention was made with government support under RO1 EB013198awarded by the National Institutes of Health. The government has certainrights in this invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:COUR-002_01US_Seqlist.txt, date recorded: Aug. 13, 2013, file size 1.17megabytes).

BACKGROUND OF INVENTION

Inflammatory diseases and disorders are conditions in which an abnormalor otherwise deregulated inflammatory response contributes to theetiology or severity of disease. Examples include autoimmune diseasessuch as type 1 diabetes and Celiac disease.

Many of these diseases are characterized by a mononuclear cellinfiltration at a site of tissue injury or other insult. Examples ofmononuclear cells that have been observed in these infiltrations includelymphocytes, especially T lymphocytes, and cells of the mononuclearphagocyte system (MPS cells) such as monocytes, macrophages, dendriticcells, microglial cells and others.

Many of the cells observed in the mononuclear cell infiltrates aresuspected of having a role in these abnormal inflammatory responses. Forexample, in diseases such as multiple sclerosis, CD4⁺ T cells are knownto play a central role in the pathologic autoimmune response. At anearlier time point in T cell activation, dendritic cells and other MPScells may be responsible for activation of CD4⁺ T cells. MPS cells couldalso contribute to inflammation through phagocytosis although in atleast some inflammatory diseases it is not clear whether such cellswould be capable of this in the absence of CD4⁺ T cells.

Peripheral blood monocytes may be classified into one of two groupsaccording to the expression or not of certain cell surface molecules. Inparticular, human “resident monocytes” or “mature monocytes” areunderstood to have a CD14^(lo)CD16⁺ phenotype (the mouse counterpart isCX₃CR1^(hi)CCR2⁻Gr1⁻). Another group of cells, the “inflammatorymonocytes” or “immature monocytes” are understood to have a CD14⁺CD16⁻phenotype (the mouse counterpart is CX₃CR1^(lo)CCR2⁺Gr1⁺). (Geissmann F.et al. 2003 Immunity 19: 71-82)

Importantly, while the latter are understood to be “inflammatory” in thesense that they are observed to migrate into inflamed tissue from bonemarrow derived peripheral blood cells, these cells have not been shownto cause inflammation either directly or through the action of othercells. Further, the various MPS cells that may be formed when thesecells differentiate have also not been shown to cause inflammation.

Conventional clinical strategies for general long-term immunosuppressionin disorders associated with an undesired immune response are based onthe long-term administration of broad acting immunosuppressive drugs,for example, signal 1 blockers such as cyclosporin A (CsA), FK506(tacrolimus) and corticosteroids. Long-term use of high doses of thesedrugs can have toxic side-effects. Moreover, even in those patients thatare able to tolerate these drugs, the requirement for life-longimmunosuppressive drug therapy carries a significant risk of severe sideeffects, including tumors, serious infections, nephrotoxicity andmetabolic disorders.

Methods of inducing antigen-specific tolerance have been developed,including cell coupling of an antigen or peptide. For example, in onemethod, peptide induced cell coupled tolerance involved collection,separation and treatment of peripheral blood cells with disease specificautoantigens and the ethylene carbodimide (ECDI) coupling reagent understerile conditions, and subsequent re-infusion into the donor/patient.This process is costly and must be conducted under closely monitoredconditions by skilled practitioners and is limited in the number ofcenters that can conduct the procedure. The use of red blood cells asthe donor cell type expands the potential source to include allogeneicdonors thus increasing the supply of source cells dramatically andpotentially expanding the delivery of this therapy to any settingcertified for blood transfusion. These approaches have significantlimitations in terms of supply of source cells and necessity for tissuetype matching to minimize immune response to the donor cells. Inaddition the local treatment of the cells to couple autoantigens viaEDCI presents a significant quality control issue. Furthermore, theseapproaches also require at least some knowledge of the pathologicalantigen for which immune tolerance is sought.

Recently, peptide-coupled particles have been described which eliminatesthe requirement for a supply of source cells and circumvents thetissue-typing requirement of the prior approaches, See WO 2010/085509,incorporated by reference herein in its entirety. Not withstanding, theuse of antigens coupled to the outside of particles is associated withincreased anaphylaxis and has significant chemistry, manufacturing andcontrol issues. Surprisingly, when the antigen is encapsulated withinthe particle, these adverse events are avoided. Even more surprisingly,the size and the charge can be altered to enhance tolerance to specificantigens.

Antigen-specific tolerance is generally not ideal because specificantigens/epitopes are generally not known in human diseases.Furthermore, antigens can vary from subject to subject in order for anantigen specific approach to be effective, therefore it would benecessary to determine which antigens each individual patient wouldrecognize, or it would require coupling a library of possible peptidesto the particles prior to administration. The synthesis and individualcoupling of these peptides is both time consuming and expensive.Therefore, a need exists for a therapy which solves both of theseproblems thereby eliminating the need to for a source of tissue matchedcells.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides compositions (e.g.,for induction of antigen-specific tolerance) comprising a carrierparticle (e.g., PLG particle) attached to an antigenic peptide. Incertain embodiments, the carrier particle is apoly(lactide-co-glycolide) (PLG) particle. In other embodiments, thecarrier particle is a PLURIONICS® stabilized polypropylene sulfideparticle.

In some embodiments, the present invention provides compositionscomprising: an antigen coupled to a carrier particle with a negativezeta potential. In some embodiments, the zeta potential of the particleis from about −100 mV to about 0 mV. In some embodiments, the zetapotential of the particle is from about −50 mV to about −40 mV. In someembodiments, the particle is a co-polymer having a molar ratio fromabout 50:50, 80:20 to about 100:0. In some embodiments, the co-polymersratio may be, but not limited to, polystyrene:poly(vinylcarboxylate)/80:20, polystyrene:poly(vinyl carboxylate)/90:10,poly(vinyl carboxylate):polystyrene/80:20, poly(vinylcarboxylate):polystyrene/90:10, polylactic acid:polyglycolic acid/80:20,or polylactic acid:polyglycolic acid/90:10, or polylacticacid:polyglycolic acid/50:50. Yet in other embodiments, the particle isa polystyrene particle, a carboxylated polystyrene particle, PLURIONICS®stabilized polypropylene sulfide particle, or a poly(lactic-co-glycolicacid) particle. In some embodiments, the particle is apoly(lactic-co-glycolic acid) particle.

In some embodiments, the particle has an average diameter of betweenabout 0.1 μm to about 10 μm. In some embodiments, the particle has anaverage diameter of between 0.2 μm and about 2 μm. In some embodiments,the particle has a diameter of between about 0.3 μm to about 5 μm. Insome embodiments, the particle has a diameter of between about 0.5 μm toabout 3 μm. In some embodiments, the particle has a diameter of betweenabout 0.5 μm to about 1 μm. In some embodiments, the particle has adiameter of about 0.5 μm.

In further embodiments, the antigen comprises at least a portion of anautoimmune antigen, an antigen expressed on a tissue to be transplantedinto a subject, an enzyme, or an allergen. In some embodiments, theantigen comprises at least a portion of myelin basic protein,acetylcholine receptor, endogenous antigen, myelin oligodendrocyteglycoprotein, pancreatic beta-cell antigen, insulin, proinsulin,islet-specific glucose-6-phosphatase catalytic subunit-related protein(IGRP), glutamic acid decarboxylase (GAD), collagen type 11, humancartilage gp39, fp130-RAPS, proteolipid protein, fibrillarin, smallnucleolar protein, thyroid stimulating factor receptor, histones,glycoprotein gp70, pyruvate dehydrogenase dehydrolipoamideacetyltransferase (PCD-E2), hair follicle antigen, aqua porin 4,Desmoglein 1, Desmoglein 3, nicotinic acetylcholine receptor, A-gliaden,and human tropomyosin isoform 5, Bahia grass pollen (BaGP), peachallergen Pru p 3, alpha s 1-Casein Milk allergen, Apig1 celery allergen,Bere1 Brazil nut allergen, B-Lactoglobulin Milk allergen, Bovine serumalbumin, Cor a 1.04 Hazelnut allergen, Ovalbumin Egg allergen, Advate,antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII Fcfusion protein, Refacto, Novo VIIa, recombinant factor VII, eptacogalfa, Helixate, Monanine, Coagulation Factor IX, Wilate, Ceredase,Alglucerase, Cerezyme, Imiglucerase, Elelso, taliglucerase alfa,Fabrazyme, Agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme,Acid-glucosidase, Elaprase, iduronate-2-sulfatase, Naglazymearylsufatase B, or N-acetylgalactosamin e-4-sulfatase, proteinaceoustherapies used in enzyme or coagluation factor replacement such asmyozyme, alglucerase, imiglucerase, taliglucerase, agalsidase beta,1-iduronidase, acid glucosidase, Iduronate-2-sulfatase,N-acetylgalactosamnie-4-sulfatase, antihemophilic factor, factor VII,eptacogalfa, factor IX, miglustat, romiplastim, epotetin alpha, proteinC, laronidase, lumizyme or Factor VIII.

In further embodiments, the antigen comprises an autoimmune antigen, anantigen expressed on a tissue to be transplanted into a subject, anenzyme, or an allergen. In non-limiting embodiments, the antigencomprises, for example, myelin basic protein, acetylcholine receptor,endogenous antigen, myelin oligodendrocyte glycoprotein, pancreaticbeta-cell antigen, insulin, glutamic acid decarboxylase (GAD), collagentype 11, human cartilage gp39, fp130-RAPS, proteolipid protein,fibrillarin, small nucleolar protein, thyroid stimulating factorreceptor, histones, glycoprotein gp70, pyruvate dehydrogenasedehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, aquaporin 4, Desmoglein 1, Desmoglein 3, nicotinic acetylcholine receptor,A-gliaden, and human tropomyosin isoform 5, Bahia grass pollen (BaGP),peach allergen Pru p 3, alpha s 1-Casein Milk allergen, Apig1 celeryallergen, Bere1 Brazil nut allergen, B-Lactoglobulin Milk allergen,Bovine serum albumin, Cor a 1.04 Hazelnut allergen, insulin, proinsulin,islet-specific glucose-6-phosphatase catalytic subunit-related protein(IGRP), Ovalbumin Egg allergen, proteinaceous therapies used in enzymeor coagluation factor replacement such as myozyme, alglucerase,imiglucerase, taliglucerase, agalsidase beta, 1-iduronidase, acidglucosidase, Iduronate-2-sulfatase, N-acetylgalactosamnie-4-sulfatase,antihemophilic factor, factor VII, eptacogalfa, factor IX, miglustat,romiplastim, epotetin alpha, protein C, laronidase, lumizyme FactorVIII.

In further embodiments, the particles are coupled to an antigencomprising one or more epitopes. In a further embodiment, the epitope isassociated with an allergy, an autoimmune disease, an enzyme used inenzyme replacement therapy, lysosomal storage disease, or aninflammatory disease or disorder. In one embodiment, the epitope isassociated with type 1 diabetes, multiple sclerosis, Systemic Lupus,Neuromyelitis Optica, Idiopathic Thrombocytopenic Purpura, ThromboticThrombocytopenic Purpura, Membranous Nephropathy, Bullous Phemphigoid,Phemphigus Vulgaris, Myasthenia Gravis, a mucopolysaccharide storagedisorder, gangliosidosis, alkaline hypophosphatasia, cholesterol esterstorage disease, hyperuricemia, growth hormone deficiency, renal anemia,Gaucher's disease, Fabry's disease, Hurler's disease, Hunter's disease,Maroteaux-Lamy disease, hemophilia A, hemophilia B, von Wilebranddisease, venous thrombosis, purpura fulminans, mucopolysaccaridosis VI,pompe disease, Celiac's disease, or inflammatory bowel disease,including Crohn's disease or colitis, e.g. ulcerative colitis. In afurther embodiment the epitopes are found within proteinaceous therapiesused in enzyme or coagluation factor replacement such as myozyme,alglucerase, imiglucerase, taliglucerase, agalsidase beta,1-iduronidase, acid glucosidase, Iduronate-2-sulfatase,N-acetylgalactosamnie-4-sulfatase, antihemophilic factor, factor VII,eptacogalfa, factor IX, miglustat, romiplastim, epotetin alpha, proteinC, laronidase, lumizyme Factor VIII. In a further embodiment, theepitope is an epitope described in Tables 2 or 3. In one embodiment, theparticles are coupled to antigens comprising only one epitope associatedwith one disease and/or disorder. In a further embodiment, antigenscomprise more than one epitope associated with the same disease and/ordisorder. In a further embodiment, the antigens comprise more than oneepitope associated with different diseases and/or disorders.

In some embodiments, the antigen is coupled to said particle by aconjugate molecule. In some embodiments, the antigen is coupled to saidparticle by a linker. In some embodiments, the conjugate molecule isethylene carbodiimide (ECDI). In certain embodiments, the antigen islinked by a streptavidin-biotin complex. In some embodiments, thelinkers can include, but are not limited to, a variety of bifunctionalprotein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particular coupling agents includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

In some embodiments, the antigen is coupled to the outside of theparticle with a negative zeta potential. In some embodiments, theantigen is encapsulated in the particle which has a negative surfacezeta potential. In some embodiments, the particle is biodegradable. Insome embodiments, the particle is surface functionalized. In someembodiments, the particle is carboxylate surface functionalized.

In some embodiments, the present invention provides methods of inducingantigen-specific tolerance in a subject comprising: administering tosaid subject an effective amount of a composition comprising anantigen-coupled particle to said subject, wherein said particle has anegative zeta potential, and wherein said particle and antigen inducetolerance of said antigen in said subject. In some embodiments,administering is performed to treat or prevent a disease or condition.In some embodiments, administering is performed prior or subsequent toonset of a disease or condition that is caused by said antigen. In someembodiments, the disease or condition is selected from the groupconsisting of: an autoimmune disease, inflammatory disease, an allergy,transplantation rejection, a lysosomal storage disease, an enzymedeficiency, inflammatory response and a hyperimmune response. In someembodiments, the disease or condition is selected from the groupconsisting of: multiple sclerosis, type 1 diabetes, asthma, a foodallergy, an environmental allergy, Celiac disease, inflammatory boweldisease, including Crohn's disease or ulcerative colitis, and acondition caused by said antigen in said subject to reduce overreactionto said antigen. In some embodiments, methods further comprise repeatingsaid administration of said composition into said subject.

In a further embodiment, the administration of the particles results inactivation induced death of effector T cells.

In a further embodiment, the administration of the particles results inanergy of effector T cells.

In a further embodiment, the administration of particles results inapoptosis of effector T cells.

In a further embodiment, the administration of particles results in theconversion of effector T cells to regulatory T cells.

In a further embodiment, the administration of particles results in theinduction and expansion of both antigen specific and non-specificregulatory T cells. In a further embodiment, the administration ofparticles results in the isolation of effector T cells in the lymphnodes and spleen inhibiting their ability to traffic to peripheral sitesand cause inflammation.

In a further embodiment, the administration of particles results in thedown regulation of T cell dependent antibody production.

In certain embodiments, the present invention provides methods fortreating celiac disease in a subject comprising administering to saidsubject an effective amount of a composition comprising anantigen-coupled particle to the subject, wherein the particle has anegative zeta potential. In certain embodiments, the antigen is gliadenor a gliaden epitope. In some embodiments, the antigen is one or moreantigens selected from the group consisting of SEQ ID NOs: 1295-1724,SEQ ID NOs: 1726-1766 and SEQ ID NOs: 4986-5140. In some embodiments,the antigen is gliaden and the antigen-coupled particle has apost-synthesis average size of about 600-1500 nanometers and apost-synthesis average charge of about −30 to about −80 mV. In someembodiments, the particle has a post-synthesis average size of about600-1200 nanometers and a post-synthesis average charge of about −40 toabout −70 mV. In certain embodiments, the particle has a post-synthesisaverage size of about 600 microns and a post-synthesis average charge ofabout −50 mV. In further embodiments, the particle is a polystyreneparticle, a carboxylated polystyrene particle, a PLURIONICS stabilizedpolypropylene sulfide particle, or a poly(lactic-co-glycolic acid)particle.

In some embodiments, the present invention provides methods of treatingdiabetes in a subject comprising administering to said subject aneffective amount of a composition comprising an antigen-coupled particleto the subject, wherein the particle has a negative zeta potential. Insome embodiments, the diabetes is type I diabetes. In some embodiments,the diabetes is type II diabetes.

In some embodiments, the antigen is insulin, proinsulin, islet-specificglucose-6-phosphatase catalytic subunit-related protein (IGRP) orepitopes derived from insulin proinsulin, or IGRP. In some embodiments,the antigen is one or more antigen selected from the group consisting ofID NOs: 1767-1840, SEQ ID NOs: 1842-1962, SEQ ID NOs: 1964-2027, SEQ IDNOs: 2029-2073, SEQ ID NOs: 2075-2113, SEQ ID NOs: 2115-2197, SEQ IDNOs: 2199-2248, SEQ ID NOs: 2250-2259, SEQ ID NOs: 2261-2420, SEQ IDNOs: 2422-2486, and SEQ ID NOs: 2489-2505. In some embodiments, antigenis insulin and the antigen-coupled particle has a post-synthesis averagesize of about 300-800 nanometers and a post-synthesis average charge ofabout −30to about −70 mV. In some embodiments, the particle has apost-synthesis average size of about 350-600 nanometers and apost-synthesis average charge of about −40 to about −60 mV. In someembodiments, the particle has a post-synthesis average size of about 500nanometers and a post-synthesis average charge of about −50 mV. In someembodiments, the antigen is pro-insulin and the antigen-coupled particlehas a post-synthesis average size of about 300-800 nanometers and apost-synthesis average charge of about −30 to about −70 mV. In certainembodiments, the particle has a has a post-synthesis average size ofabout 400-600 nanometers and a post-synthesis average charge of about−40 to about −60 mV. In some embodiments, the particle has apost-synthesis average size of about 570 nanometers and a post-synthesisaverage charge of about −45. In some embodiments, the antigen is IGRPand the antigen-coupled particle has a post-synthesis average size ofabout 300-800 nanometers and a post-synthesis average charge of about−30 to about −70 mV. In some embodiments, the particle has a has apost-synthesis average size of about 400-700 nanometers and apost-synthesis average charge of about −40 to about −60 mV. In someembodiments, the particle has a post-synthesis average size of about 600nanometers and a post-synthesis average charge of about −40. In certainembodiments, the particle is a polystyrene particle, a carboxylatedpolystyrene particle, a PLURIONICS stabilized polypropylene sulfideparticle, or a poly(lactic-co-glycolic acid) particle.

In some embodiments, the present invention provides methods of treatinga subject undergoing enzyme replacement therapy, comprisingadministering to said subject an effective amount of a compositioncomprising an antigen-coupled particle to the subject, wherein theparticle has a negative zeta potential. In some embodiments, the subjectis undergoing enzyme replacement therapy for treatment of a diseaseselected from the group consisting of Hemophilia, Hemophilia A,Hemophilia B, von Willebrand disease, a mucopolysaccharide storagedisorder, gangliosidosis, alkaline hypophosphatasia, cholesterol esterstorage disease, hyperuricemia, growth hormone deficiency, renal anemiaGaucher's Disease, Fabry's Disease, Hurler's Disease, Pompe's Disease,Hunter's Disease, and Maroteaux-Lary Disease. In some embodiments theantigen coupled particle comprises one or more enzyme selected from thegroup consisting of Advate, antihemophilic factor, Kogenate, Eloctate,recombinant factor VIII Fc fusion protein, Refacto, Novo VIIa,recombinant factor VII, eptacog alfa, Helixate, Monanine, CoagulationFactor IX, Wilate, Ceredase, Alglucerase, Cerezyme, Imiglucerase,Elelso, taliglucerase alfa, Fabrazyme, Agalsidase beta, Aldurazyme,-I-iduronidase, Myozyme, Acid-glucosidase, Elaprase,iduronate-2-sulfatase, Naglazyme arylsufatase B, andN-acetylgalactosamin e-4-sulfatase. In some embodiments, the particle isa polystyrene particle, a carboxylated polystyrene particle, aPLURIONICS stabilized polypropylene sulfide particle, or apoly(lactic-co-glycolic acid) particle. In certain embodiments, theparticle is a co-polymer having a molar ratio from about 80:20 to about100:0. In certain embodiments, the particle is a polystyrene particle, acarboxylated polystyrene particle, a PLURIONICS stabilized polypropylenesulfide particle, or a poly(lactic-co-glycolic acid) particle. In otherembodiments, the particle is a poly(lactic-co-glycolic acid) particleand has a copolymer ratio of about 50:50 polylactic acid:polyglycolicacid. In some embodiments, the particle is a poly(lactic-co-glycolicacid) particle and has a copolymer ratio of about 50:50 polylacticacid:polyglycolic acid.

In a further embodiment, the administration of the particles of theinvention prevents the accumulation of neutrophils and othergranulocytes in a subject. In a further embodiment, the particles of theinvention are administered to a subject who has cancer.

In one embodiment, administration of the particles of the inventionincreases regeneration of damaged tissue. In a further embodiment, theparticles increase regeneration of epithelial cells. In yet a furtherembodiment, the particles increase remyelination of neurons. In anotherembodiment, the subject has an autoimmune disease. In yet anotherembodiment, the subject has inflammatory bowel disease, includingulcerative colitis, and/or Crohn's disease. In yet another embodiment,the subject has multiple sclerosis.

In some embodiments the composition is administered intravenously. Insome embodiments, the composition is administered subcutaneously,orally, intramuscularly, intra-lymphatically, portally or via aerosol.In one embodiment, administration of the negatively charged particlesinduces antigen-specific tolerance in a subject. In one embodiment, theparticles that induce antigen-specific tolerance comprise one or moreepitopes associated with an allergy, autoimmune disease, and/orinflammatory disease. In one embodiment, the epitopes are selected fromthose described in Tables 2 or 3. In one embodiment, the negativelycharged particles are polystyrene, diamond, PLURONICS® stabilizedpolypropylene sulfide, or poly(lacti-co-glycolic acid) particles. In oneembodiment the particles are carboxylated. In one embodiment, theparticles have a zeta potential of less than about −100 mV. In oneembodiment, the particles have a zeta potential between about −75 mV and0 mV, for example, between −50 mV and 0 mV, or between −100 mV and −50mV or between −75 mV and −50 mV or between −50 mV and −40 mV. In oneembodiment, the particle has an average diameter of about 0.1 μm toabout 10 μm, for example from about 0.2 μm to about 2 μm or about 0.3 μmto about 5 μm, or 0.5 μm to about 3 μm or about 0.5 μm to about 1 μm.

In one embodiment, the subject has an autoimmune disease. In oneembodiment, the autoimmune disease is multiple sclerosis, scleroderma,type-I diabetes, rheumatoid arthritis, thyroiditis, systemic lupuserythmatosis, Reynaud's syndrome, Sjögren's syndrome, autoimmuneuveitis, autoimmune myocarditis, inflammatory bowel disease, AmyotrophicLateral Sclerosis (ALS), Systemic Lupus, Neuromyelitis Optica,Idiopathic Thrombocytopenic Purpura, Thrombotic ThrombocytopenicPurpura, Membranous Nephropathy, Bullous Phemphigoid, PhemphigusVulgaris, Myasthenia Gravis, Celiac disease, ulcerative colitis, orCrohn's disease. In one embodiment, the particle comprises a full-lengthpolypeptide or fragment thereof. In one embodiment, the particlecomprises one or more myelin basic protein epitopes. In one embodiment,the myelin basic protein epitope is from SEQ ID NO: 4975 or SEQ ID NO:4976. In one embodiment, the particles comprise one or more myelinoligodendrocyte glycoprotein epitopes. In one embodiment, the myelinoligodendrocyte glycoprotein epitope is from SEQ ID NO: 1 or SEQ ID NO:4978. In one embodiment, the particle contains one or more insulinepitopes. In one embodiment, the one or more insulin epitopes is fromSEQ ID NO: 4981. In one embodiment, the particle comprises one or moreglutamic acid decarboxylase epitopes. In one embodiment, the glutamicacid decarboxylase epitopes is from SEQ ID NO: 4982. In one embodiment,the particle contains one or more proteolipid protein epitopes. In oneembodiment, the proteolipid protein epitope is from SEQ ID NO: 4977. Inone embodiment, the particle comprises one or more gliaden epitopes. Inone embodiment, the gliaden epitopes comprise SEQ ID NOs: 4983-4985.

In some embodiments, the present invention further provides a processfor the preparation an immune modified particle with a negative zetapotential said process comprising: contacting an immune modifiedparticle precursor with a buffer solution under conditions effective toform the immune modified particle with a negative zeta potential. Insome embodiments, the immune modified particle precursor is formed byco-polymerization. In some embodiments, the buffer solution has a basicpH. In some embodiments, buffer solution is sodium bicarbonate,potassium bicarbonate, lithium bicarbonate, potassium dihydrogenphosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate.

In some embodiments, the present invention provides a compositioncomprising an antigen encapsulated within the core of asurface-functionalized liposome. In a further embodiment, the liposomeis composed at a 30:30:40 ratio ofphosphatidylcholine:phosphatidylglycerol:cholesterol. In yet a furtherembodiment, said antigen comprises an autoimmune antigen, an antigenexpressed on a tissue to be transplanted into a subject, or an allergen.In some embodiments, the particle is a poly(lactic-co-glycolic acid)particle and has a copolymer ratio of about 50:50 polylacticacid:polyglycolic acid. In some embodiments, the particles comprisePEMA. In some embodiments, the PEMA is present at about 0.1% to about2.0%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show (FIG. 1A) a micrograph of a poly(lactide-co-glycolide)(PLG) particle. FIG. 1B and FIG. 1C show the characterization ofsurface-functionalized poly(lactide-co-glycolide) particles by dynamiclight scattering analysis, including the size distribution, average size(nm), potential (mV), and peptide coupling efficiency (%) of OVA₃₂₃₋₃₃₉and PLP₁₃₉₋₁₅₁ peptides with PLG-PEMA particles. Surface-functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 2.5×10⁵ counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles varied by5-15% per batch but generally had a Z-average diameter of 567 nm, a peakdiameter of 670 nm and a polydispersity index of 0.209.

FIG. 2 shows that PLG nanoparticles induce antigen-specific tolerance.The immunodominant proteolipid protein PLP₁₃₉₋₁₅₁ epitope(PLG-PLP₁₃₉₋₁₅₁) was used to induce tolerance for prevention ofRelapsing Experimental Autoimmune Encephalitis (R-EAE). Mice weretreated with either PLP₁₃₉₋₁₅₁-PLGA (N=5), OVA₃₂₃₋₃₃₉-PLGA (N=5), orunconjugated PLGA (N=5) on day −7 relative to the time of immunization(day 0). Peak disease was typically observed around day 12 to 14, andmice are scored for clinical disease. Particles without peptide, ormodified with the control peptide OVA₃₂₃₋₃₃₉ did not prevent diseaseinduction. However, PLGA particles modified with PLP₁₃₉₋₁₅₁ produced aclinical score of 0 (no disease) at all except low clinical scores ofunder 1 exhibited between days 20 and 30.

FIGS. 3A-3B show that the type of particle administered has an effect onthe development of EAE in the mouse model. (FIG. 3A) shows the meanclinical score and FIG. 3B) shows the mean cumulative score of the EAEanimals. Mice were treated with either OVA₃₂₃₋₃₃₉-PLS (N=5),OVA₃₂₃₋₃₃₉-PLGA_(PHOSPOREX) (N=5), OVA₃₂₃₋₃₃₉-PLGA_(PEMA) (N=5),PLP₁₃₉₋₁₅₁-PLA (N=5), PLP₁₃₉₋₁₅₁-PLGA_(PHOSPOREX) (N=5), orPLP₁₃₉₋₁₅₁-PLG_(PEMA) (N=5) on day −7 relative to the time ofimmunization (day 0). Peak disease was typically observed around day 12to 14, and mice are scored for clinical disease. Particles, of anycomposition that were modified with the control peptide OVA₃₂₃₋₃₃₉ didnot prevent disease induction. However, the PLP₁₃₉₋₁₅₁ coupled PLG beadswere more effective in down-regulating induction of R-EAE thanPLP₁₃₉₋₁₅₁ coupled commercial (phosphorex) PLG or polystyrene.

FIG. 4 shows that those mice treated with soluble OVA on day 28exhibited a decrease in temperature compared with those animals treatedwith the OVA-PLG particle. No decrease in body temperature was observedwithin 1 hour of delivering the particles.

FIGS. 5A-5B show that administration of PLP-PLG during remission doesnot result in any anaphylaxis-associated mortality. EAE was induced insix to eight week old female SJL/J mice by subcutaneous injection ofPLP₁₃₉₋₁₅₁ in CFA, and development of clinical disease was monitored andrecorded (FIG. 5B). On day 21 relative to disease induction, mice weregiven iv injections of soluble PLP₁₃₉₋₁₅₁ (clear squares), solubleOVA₃₂₃₋₃₃₉ (clear circles), or the same peptides coupled to PLGnanoparticles (solids). Temperature of animals was monitored andrecorded every 10 minutes for 1 hour following injection (FIG. 5A)

FIGS. 6A-6F show the optimal dosing of PLP₁₃₉₋₁₅₁-PLG administeredintravenously seven days prior to disease induction. Development ofclinical disease was measured in comparison to SJL/J mice treated withOVA₃₂₃₋₃₃₉-PLG (FIG. 6A). Six to eight week old female SJL/J mice wereinjected iv with either PLP₁₃₉₋₁₅₁ (square)- or OVA₃₂₃₋₃₃₉(circle)-coupled PLG nanoparticles. EAE was induced by subcutaneousinjection of PLP₁₃₉₋₁₅₁ in CFA 7 days (FIG. 6B), 25 days (FIG. 6C), or50 days (FIG. 6D) later. Animals from panel B were followed for clinicaldisease for 100 days. On day 8 relative to disease induction, adelayed-type hypersensitivity (DTH) reaction was carried out in a subsetof the mice shown in panel B (FIG. 6E). Selected representative animalsfrom the PLP₁₃₉₋₁₅₁/CFA primed groups in panel B (OVA₃₂₃₋₃₃₉-PLG andPLP₁₃₉₋₁₅₁-PLG) were ear-challenged with the priming PLP₁₃₉₋₁₅₁ epitopeand the OVA₃₂₃₋₃₃₉ control peptide. Ear swelling as a measure of DTH wasdetermined 24 h later and responses prior to challenge were subtracted.Six to eight-week old female SJL/J mice were injected intravenously withPLP₁₇₈₋₁₉₁ (triangle)-, OVA₃₂₃₋₃₃₉ (circle), or PLP₁₃₉₋₁₅₁(square)-coupled PLG nanoparticles, or with uncoupled particles alone(outlined circle) (FIG. 6F). EAE was induced 7 days afterward bysubcutaneous injection of PLP₁₇₈₋₁₉₁ in CFA, and disease was monitoredat the time points shown.

FIGS. 7A-7D show prophylactic tolerance is most efficient when thePLG-PLP₁₃₉₋₁₅₁ particles are administered either intravenously orintraperitoneally. Animals treated with PLP₁₃₉₋₁₅₁-PLG administeredintravenously did not develop disease and had mean clinical scores of 0at most time points.

FIGS. 8A-8F show that the administration of OVA₃₂₃₋₃₃₉-PLG particlesinhibited the Th1 and Th17 responses in the treated animals.

FIGS. 9A-9C show a reduction in immune cell infiltration within thespinal cord of animals treated with PLP₁₃₉₋₁₅₁-PLG that and was moresimilar to native tissue than to tissue from OVA₃₂₃₋₃₃₉-PLG treatedanimals. OVA₃₂₃₋₃₃₉-PLG treated animals had positive staining for CD45,CD4, and CD11b; whereas, PLP₁₃₉₋₁₅₁-PLG treated animals had minimalstaining for these factors.

FIGS. 10A-10C show that administration of PLP₁₃₉₋₁₅₁-PLG particlesinhibits blood brain barrier (BBB) disruption and macrophage activationin the spinal cord of treated mice. Animals were treated with eitherComplete Freund's Adjuvant (CFA), OVA₃₂₃₋₃₃₉ PLG particles, orPLP₁₃₉₋₁₅₁-PLG particles. The clinical scores and percent incidence ofEAE were determined (FIG. 10B) and the spinal cords observed via in vivoimaging (FIG. 10A and FIG. 10C).

FIGS. 11A-11F show the spinal cords of treated mice via in vivo imaging.FIGS. 11C-F are graphs showing the quantification of the image data.

FIG. 12 shows that the administration of PLG particles in whichPLP₁₃₉₋₁₅₁ has been encapsulated inhibits the induction of R-EAE inmice. The ability to encapsulate autoantigens allows for the used ofcomplex mixtures of proteins or even organ homogenates not possible withsurface binding, allowing for more antigen coverage and thus moreeffectively deal with epitope spreading.

FIG. 13 shows that animals treated with the PLP₁₃₉₋₁₅₁-PLG particles andan anti-CD25 antibody demonstrated, at times, a greater mean clinicalscore than those animals treated with PLP₁₃₉₋₁₅₁-PLG particles and acontrol IgG antibody.

FIGS. 14A-14E show that therapeutic tolerance induced by PLP₁₃₉₋₁₅₁-PLGparticles in active and adoptive EAE. Adoptive EAE was induced in six toeight-week old female SJL/J mice by adoptive transfer of 2.5×10⁶PLP₁₃₉₋₁₅₁-activated blasts. Mice were injected iv with PLP₁₃₉₋₁₅₁(squares) or OVA₃₂₃₋₃₃₉ (circles) peptide coupled to 500 nm PLGnanoparticles 2 days (FIG. 14A), 14 days (FIG. 14C), 18 days (FIG. 14E),or 21 days (FIG. 14F) following disease induction. Clinical diseasescores were compared to those following treatment with antigen-coupledsplenocytes (FIG. 14A). Brain and spinal cord were collected fromPLP₁₃₉₋₁₅₁- or OVA₃₂₃₋₃₃₉-tolerized mice for histological analysis onday 42. Sections from mice from panel A were stained for PLP protein andCD45 (FIG. 14B). Spinal cord sections from mice from panel (FIG. 14C)were stained with Luxol Fast Blue (FIG. 14D). Areas of demyelination andcellular infiltration are indicated by arrows.

FIGS. 15A-15B show graphs depicting the mean clinical scores of micewith active EAE and adoptive EAE after treatment with either SP or PLGparticles conjugated to OVA₃₂₃₋₃₃₉ or PLP₁₃₉₋₁₅₁. Mice were injected ivwith PLP₁₃₉₋₁₅₁-SP, PLP₁₃₉₋₁₅₁-PLG, or OVA₃₂₃₋₃₃₉-SP, or OVA₃₂₃₋₃₃₉-PLGpeptide coupled to 500 nm nanoparticles 10 days (FIG. 15A) or 2 days(FIG. 15B) following disease induction and the mean clinical score wasdetermined. In both cases, administration of PLP₁₃₉₋₁₅₁-PLG particlesinduces tolerance in the mice.

FIGS. 16A-16H show the infiltration of central nervous system immunecells is also drastically reduced in PLP-PLG tolerized mice. SJL/J micewere injected i.v. with 500 nm PLG nanoparticles coupled with PLP₁₃₉₋₁₅₁(squares) or OVA₃₂₃₋₃₃₉ (circles) 2 days following EAE induction byadoptive transfer. At the peak of disease (day 14) brains and spinalcords were removed and the number of lymphocytes (FIG. 16B), APCs (FIG.16C), microglia (FIG. 16D), peripheral dendritic cells (FIG. 16E),myeloid dendritic cells (FIG. 16F) and macrophages (FIG. 16G) wereenumerated by flow cytometry. The gating strategy for these populationsis depicted in (FIG. 16A). CNS cell preparations were stimulated withPMA and ionomycin for 5 h prior to intracellular staining for IL-17A andIFN-γ (H).

FIG. 17 shows that administration of the PLP₁₃₉₋₁₅₁ peptide encapsulatedin a PLG particle induces tolerance when the particle is administeredwith PBS. However, administration of the anti-PD-1 antibody decreasesthis tolerance.

FIG. 18 shows that administration of the PLP₁₃₉₋₁₅₁ peptide encapsulatedin a PLG particle induces tolerance when the particle is administeredwith PBS. Administration of an anti-CD40 antibody decreases thistolerance, but this decrease in tolerance is reversed by the addition ofan anti-IL-12 antibody.

FIGS. 19A-19G show that the prophylactic administration of OVA-PLGdecreased the secretion of IL-4, IL-5, IL-13 and IL-10, and reduced thelevels of serum OVA IgE and eosinophils in the lung.

FIGS. 20A-20B show that OVA encapsulated in PLG particlesprophylactically inhibits OVA-specific in vitro recall responses frommediastinal lymph nodes. The lymph node proliferation observed afterrestimulation with 25 μg OVA is decreased in those animals treated withOVA-PLG (FIG. 20A). Moreover treatment with OVA-PLG decreases therelease of cytokines after restimulation with OVA. Levels of IL-4, IL-5,IL-13, and IL-10 are decreased in mice treated with OVA-PLG (FIG. 20B).

FIGS. 21A and 21B show that the therapeutic administration of OVA-PLGdecreased the secretion of IL-4, IL-5, IL-13 and IL-10, and reduced thelevels of serum OVA IgE and eosinophils in the lung.

FIGS. 22A-22B show that OVA encapsulated in PLG particlestherapeutically downregulates OVA-Specific Th2 Cytokines in the BALfluid better than OVA-coupled PLG particles. Mice were treatedintraperitoneally with OVA/Alum at a dose of 10 μg/mouse on day 0 andday 14. The mice were intravenously administered with either OVA-coupledto PLG particles or OVA encapsulated in PLG particles on days 28, and42. Between days 56-58, the mice were treated three times withaerosolized OVA. The graphs depict cytokine secretion when the animalswere treated with either OVA coupled to PLG particles (FIG. 22A) or OVAencapsulated within PLG particles (FIG. 22B).

FIGS. 23A-23C show the blood glucose levels of type 1 diabetic animalsafter treatment with p31-PLG particles. Administration of the p31peptide coupled PLG particles resulted in lower blood glucose levelscompared to those seen after administration with the MOG₃₅₋₅₅ peptidecoupled particles (FIG. 23A and FIG. 23B). The percent of IFNγ secretingcells observed in the animals was also reduced in the p31-PLG treatedmice compared with the MOG₃₅₋₅₅ peptide-PLG treated mice (FIG. 23C).

FIGS. 24A-24B show p31-PLG induced tolerance requires Tregs. Type 1diabetes was induced in mice by adoptive transfer. Two hours after theactivated cells were transferred to the NOD.SCID mice, the mice weretolerized with either p31-PLG or MOG₃₅₋₅₅ PLG particles. Depletion ofTregs abrogates the tolerance induced by administration of p31-PLGparticles.

FIG. 25 shows administration of insulin coupled PLG particlessignificantly increased the percentage of mice that did not developdiabetes over 300 days (69.6% compared to 22.7%; p=0.0027). NOD micewere treated with either BSA (N=22) or insulin (N=23) coupled PLGparticles via intravenous administration at 6, 8, and 10 weeks of age.The mice were then assayed for the development of diabetes.

FIG. 26 shows the percent of CD45.1 donor cells observed in therecipient mice. Female CD45.2 mice were tolerized with either OVA-PLG orDby-PLG on day −7. On day −1, the mice were irradiated with 200 rads andwere then transplanted with 1×10⁶, 5×10⁶, or 1×10⁷ bone marrow cellsfrom male CD45.1 mice on day 0. The recipient mice were then tolerizedwith either OVA-PLG, Dby-SP, or Dby-PLG on day 1 and the blood harvestedfor FACS analysis of chimerism.

FIG. 27 shows the percent of donor CD45.1 cells in the recipient miceafter tolerization with either OVA-PLG, Dby-SP, or Dby-PLG on day 1. Onepositive control mouse did not demonstrate significant engraftment(˜10%). All negative control mice did not engraft donor cells. OneDby-SP mouse did not demonstrate significant engraftment (˜10%). TwoOVA-PLG mice engrafted donor cells (˜10%): one completely rejected byWeek 16. One Dby-PLG mouse started to reject at Week 12 and was at 10%by Week 16. The Dby-PLG group ranged from 10%-56% engraftment by Week16. The OVA-PLG mice demonstrated: 1) Spontaneous engraftment, 2)Sequence homology between OVA323 and Dby, or 3) tolerogenic propertiesof particles. Dby-PLG allows for more engraftment than Dby-SP andOVA-PLG.

FIG. 28 shows that the timing tolerance has an effect on the percent ofCD45.1 cells in the recipient mouse. Positive Controls show lessengraftment (˜4%) than expected (˜10%). One Negative control mouse had5% engraftment Out of all 3 OVA-PLG groups, one mouse in the Day −7, Day+1 group showed engraftment (12%). Tolerance on day 1 is more clinicallyrelevant than tolerance on day −7.

FIG. 29 shows that coumarin-6 PLGA particles, that were either coupledto an antigen or were antigen-free, were detectable at 3 hourspost-administration, but not at 24 hours post-administration. Theparticles were detectable at 3 hours post-administration, but not at 24hours post-administration. Naïve uninjected mouse (top row) as comparedto i.v. fluorescent PLGA/PEMA microparticle injected mouse spleen (leftcolumn), liver (middle column) and lung (left column) sections at3-hours post injection (middle row) and 24-hours (bottom row)post-injection, counterstained with DAPI.

FIG. 30 shows that PLGA particles co-localized after 6 and 15 hours withF4/80⁺ cells in the liver.

FIG. 31 shows that marginal zone macrophages predominantly uptakeTAMRA-labeled PLP₁₃₉₋₁₅₁-coupled particles 24 hours after intravenousinfusion. The highest percentage of PLP₁₃₉₋₁₅₁+ cells are marginal zonemacrophages.

FIG. 32 depicts the daily mean clinical score against the number of daysPLP₁₃₉₋₁₅₁/CFA priming. PLP₁₃₉₋₁₅₁/CFA-induced R-EAE is inhibited inSJL/J mice by the induction of immunological tolerance usingsurface-functionalized poly(lactide-co-glycolide) particles containingsoluble PLP₁₃₉₋₁₅₁ within their cores.

FIG. 33 shows that mice treated with encapsulated OVA-PLG showed thegreatest reduction in eosinophil accumulation.

FIG. 34 shows that mice treated with encapsulated OVA-PLG showed thegreatest reduction in serum IgE levels compared to untreated or controltreated animals.

FIG. 35 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble PLP₁₃₉₋₁₅₁within their cores by dynamic light scattering analysis.Surface-functionalized poly(lactide-co-glycolide) particles wereanalyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments,Westborough, Mass.) at a count rate of 1.792×105 counts per second in18.2 MΩ water. The population of surface-functionalizedpoly(lactide-co-glycolide) particles had a Z-average diameter of 584 nm,a peak diameter of 679 nm and a polydispersity index of 0.162. Theseresults are representative of 6 batches of syntheses, following theprotocol written above.

FIG. 36 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble PLP₁₃₉₋₁₅₁within their cores by ζ-potential measurement. Surface functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 6.67×104 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had a peakζ-potential of −48.9 mV and a deviation of 5.14 mV. These results arerepresentative of 6 batches of syntheses, following the protocol writtenabove.

FIG. 37 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble ovalbumin withintheir cores by dynamic light scattering analysis. Surface-functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 1.822×105 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had aZ-average diameter of 569.7 nm, a peak diameter of 700.3 nm and apolydispersity index of 0.230. These results are representative of 3batches of syntheses, following the protocol written above.

FIG. 38 shows characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble ovalbumin withintheir cores by ζ-potential measurement. Surface functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 2.67×104 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had a peakζ-potential of −52.2 mV and a deviation of 5.38 mV. These results arerepresentative of 3 batches of syntheses, following the protocol writtenabove.

FIG. 39 shows a graph demonstrating that surface-functionalizedliposomes containing soluble PLP₁₃₉₋₁₅₁ peptide within their coresinduce immunological tolerance in the murine model of multiplesclerosis. Animals were treated with either surface-functionalizedliposomes containing soluble PLP₁₃₉₋₁₅₁ peptide within their cores(circles) or surface-functionalized liposomes containing solubleOVA₃₂₃₋₃₃₉ peptide (squares). The mean clinical scores of those animalsreceiving the PLP₁₃₉₋₁₅₁ peptide liposomes was lower than that ofanimals receiving the OVA₃₂₃₋₃₃₉ peptide liposomes.

FIGS. 40A-40B show that the charge of the particle administered has aneffect on the development of EAE in the mouse model. FIG. 40A shows themean clinical score, and FIG. 40B shows the mean cumulative score of theEAE animals. Mice received TIMP (tolerogenic immune modifying particles)having a charge of either −60 mv or −25 mv conjugated to an antigen.Mice were treated with either OVA₃₂₃₋₃₃₉-TIMP-60 mv, OVA₃₂₃₋₃₃₉-PLGA-25mv, PLP₁₃₉₋₁₅₁-TIMP-60 mv, or PLP₁₃₉₋₁₅₁-PLGA-25 mv and scored forclinical disease. The more negatively charged particles, TIMP_(−60mv),induce tolerance more effectively than the PLGA-25 mv particles.

FIGS. 41A-41B show that the charge of the immune-modifying particle isimportant for targeting the immune modifying particle to the antigenpresenting cell. Wild type or MARCO−/+ animals were treated with eitherPS-IMP or vehicle. The results indicate that particles with a reducednegative charge have a lower efficacy because there is less interactionwith the scavenger receptor MARCO (FIG. 41A). AntiMARCO antibody aloneis not capable of providing similar efficacy at PLGA IMP (FIG. 41B).

FIGS. 42A-42B demonstrate the key particle parameters required fortolerance in the EAE murine model. FIG. 42A shows that the mosteffective average particle size is 500 nm. Mice were treated with either500 nm OVA₃₂₃₋₃₃₉-PSB, 100 nm PLP₁₃₉₋₁₅₁-PSB, 500 nm PLP₁₃₉₋₁₅₁-PSB,1.75 μm PLP₁₃₉₋₁₅₁-PSB, or 4.5 μm PLP₁₃₉₋₁₅₁-PSB and scored for clinicaldisease. FIG. 42B shows that 24 hours after i.v. infusion fluorescentlylabelled particles with a 50:50 lactide:glycolide ratio havesignificantly cleared from the spleen, liver and lung.

FIGS. 43A-43C demonstrate that TIMPs with encapsulated antigens aresuperior to peptide-coupled particles. In the murine allergy model,animals were exposed to OVA as an allergen, and were then treated witheither a sham-PLG, no treatment, a PLGA particle with OVA coupled to theoutside of the particle (FIG. 43A) or a PLGA particle with OVAencapsulated within the particle (TIMP) (FIG. 43B). FIG. 43A shows thatOVA-PLG surface coupled particles fail to reduce the TH2 response. FIG.43B shows that TIMP_(PEMA-60mv) (OVA encapsulated within the particle)inhibit the TH2 response. FIG. 43C shows that TIMP_(PEMA-60mv) (OVAencapsulated within the particle) inhibit recall responses.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that nanoparticles coupled to anantigen can induce tolerance to autoimmune disease and decrease theimmune response. These particles can induce tolerance regardless ofwhether they are bound to the surface of the particle or encapsulatedwithin. These, particles, therefore, may be useful in the treatment ofany disease or condition characterized by an excessive inflammatoryimmune response, such as autoimmune diseases or allergies.

“Particle” as used herein refers to any non-tissue derived compositionof matter, it may be a sphere or sphere-like entity, bead, or liposome.The term “particle”, the term “immune modifying particle”, the term“carrier particle”, and the term “bead” may be used interchangeablydepending on the context. Additionally, the term “particle” may be usedto encompass beads and spheres.

“Negatively charged particle” as used herein refers to particles whichhave been modified to possess a net surface charge that is less thanzero.

“Carboxylated particles” or “carboxylated beads” or “carboxylatedspheres” includes any particle that has been modified to contain acarboxyl group on its surface. In some embodiments the addition of thecarboxyl group enhances phagocyte/monocyte uptake of the particles fromcirculation, for instance through the interaction with scavengerreceptors such as MARCO. Carboxylation of the particles can be achievedusing any compound which adds carboxyl groups, including, but notlimited to, Poly(ethylene-maleic anhydride) (PEMA).

“Antigenic moiety” as used herein refers to any moiety, for example apeptide, that is recognized by the host's immune system. Examples ofantigenic moieties include, but are not limited to, autoantigens,enzymes, and/or bacterial or viral proteins, peptides, drugs orcomponents. Without being bound by theory, while the carboxylated beadsthemselves may be recognized by the immune system, the carboxylatedbeads with nothing more attached thereto are not considered an“antigenic moiety” for the purposes of the invention.

“Naked beads” or “naked particles” or “naked spheres” as used hereinrefers to beads, particles or spheres that have not been carboxylated.

“Pro-inflammatory mediators” or “pro-inflammatory polypeptides” as usedherein refers to polypeptides or fragments thereof which induce,maintain, or prolong inflammation in a subject. Examples ofpro-inflammatory mediators include, but are not limited to, cytokinesand chemokines.

As used herein, the term “Inflammatory monocyte” refers to any myeloidcell expressing any combination of CD14/CD26 and CCR2.

As used herein, the term “inhibitory neutrophil” refers to neutrophils,and/or monocyte derived suppressor cells.

As used herein, the term “Th cell” or “helper T cell” refers to CD4⁺cells. CD4⁺ T cells assist other white blood cells with immunologicprocesses, including maturation of B cells into plasma cells and memoryB cells, and activation of cytotoxic T cells and macrophages. T cellsbecome activated when they are presented with peptide antigens by MHCclass II molecules, which are expressed on the surface ofantigen-presenting cells (APCs).

As used herein, the term “Th1 cell” refers to a subset of Th cells whichproduce proinflammatory mediators. Th1 cells secrete cytokines tofacilitate immune response and play a role in host defense againstpathogens in part by mediating the recruitment of neutrophils andmacrophages to infected tissues. Th1 cells secrete cytokines includingIFN-gamma, IL2, IL-10, and TNF alpha/beta to coordinate defense againstintracellular pathogens such as viruses and some bacteria.

As used herein, the term “Th2 cell” refers to a subset of Th cells thatmediate the activation and maintenance of the antibody-mediated immuneresponse against extracellular parasites, bacteria, allergens, andtoxins. Th2 cells mediate these functions by producing various cytokinessuch as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25) that areresponsible for antibody production, eosinophil activation, andinhibition of several macrophage functions, thus providingphagocyte-independent protective responses.

As used herein, the term “Th17 cell” refers to a subset of Th cells.Th17 cells secrete cytokines to facilitate immune response and play arole in host defense against pathogens by mediating the recruitment ofneutrophils and macrophages to infected tissues. TH17 cells secretecytokines such as IL17, IL21, IL22, IL24, IL26 and TNF alpha tocoordinate defense against extracellular pathogens including fungi andbacteria.

“Coupled” as used herein refers to an antigen fixed to the outside of aparticle or encapsulated within a particle. Thus, an antigen coupled toa particle includes both surface coupling as well as encapsulationwithin the particle.

The term “IMP” as used herein refers to immune-modifying particles whichare not coupled to an antigen. The term “TIMP” as used herein refers totolerizing immune modifying particles which are coupled to an antigen.In some embodiments, the antigen is attached to the surface of the TIMP.In other embodiments, the antigen is encapsulated within the TIMP.

The particle may have any particle shape or conformation. However, insome embodiments it is preferred to use particles that are less likelyto clump in vivo. Examples of particles within these embodiments arethose that have a spherical shape.

Another aspect of the invention relates to a composition which comprisesan immune modified particle having a negative zeta potential and freefrom antigenic moieties. In a further embodiment, the invention providescompositions comprising an immune modified particle with a negative zetapotential coupled to an antigen. In a further embodiment, the antigen iscoupled to the outside of the particle. In a preferred embodiment, theantigen is encapsulated within the particle.

Yet another aspect of the invention relates to a process for thepreparation an immune modified particle with a negative zeta potentialand free from antigenic moieties. The process involves contacting animmune modified particle precursor with a buffer solution underconditions effective to form the immune modified particle with anegative zeta potential. In some embodiments of this invention, theimmune modified particle precursor is formed via co-polymerization. Theparticle microstructure may depend on the method of co-polymerization.

In some embodiments, an antigenic peptide molecule is coupled to thecarrier particle (e.g. immune modified particle) by a conjugate moleculeand/or linker group. In some embodiments, coupling of the antigenicpeptide and/or apoptotic signalling molecule to the carrier (e.g., PLGparticle) comprises one or more covalent and/or non-covalentinteractions. In some embodiments, the antigenic peptide is attached tothe surface of the carrier particle with a negative zeta potential. Insome embodiments, the antigenic peptide is encapsulated within thecarrier particle with a negative zeta potential.

In one embodiment, the buffer solution contacting the immune modifiedparticle may have a basic pH. Suitable basic pH for the basic solutioninclude 7.1, 7.5, 8.0, 8.5, 9.5, 10.0 10.5, 11.0, 11.5, 12.0, 12.5,13.0, and 13.5. The buffer solution may also be made of any suitablebase and its conjugate. In some embodiments of the invention, the buffersolution may include, without limitation, sodium bicarbonate, potassiumbicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodiumdihydrogen phosphate, or lithium dihydrogen phosphate and conjugatesthereof.

In one embodiment of the invention, the immune modified particlescontain co-polymers. These co-polymers may have varying molar ratio.Suitable co-polymer ratio of present immune modified particles may be25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30,75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12,89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or100:0. In another embodiment, the co-polymer may be periodical,statistical, linear, branched (including star, brush, or combco-polymers) co-polymers. In some embodiments, the co-polymers ratio maybe, but not limited to, polystyrene:poly(vinyl carboxylate)/80:20,polystyrene:poly(vinyl carboxylate)/90:10, poly(vinylcarboxylate):polystyrene/80:20, poly(vinylcarboxylate):polystyrene/90:10, polylactic acid:polyglycolic acid/50:50,polylactic acid:polyglycolic acid/80:20, or polylactic acid:polyglycolicacid/90:10.

In one embodiment, the particles of the invention are made by adding acomposition comprising the polymer (e.g. PLGA) to a solution ofPoly(ethylene-maleic anhydride) (PEMA). The concentration of PEMA in thesolution can be between about 0.1% and about 10%. In one embodiment, theconcentration of PEMA in the solution is between about 0.2% and about5%. In another embodiment, the concentration of PEMA in the solution isbetween about 0.1% and 4%. In another embodiment, the concentration ofPEMA in the solution is between about 0.1% and 2%. In anotherembodiment, the concentration of PEMA in the solution is between about0.5% and 1%. In one embodiment, the percentage of PEMA in solution is0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%,3%, 3.5%, 4%, 4.5%, 5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.In one embodiment, the percentage of PEMA in the solution is about 0.5%.In another embodiment, the percentage of PEMA in the solution is about1.0%. Other compounds that may be used include, but are not limited to,Poly(ethylene-alt-maleic anhydride), Poly(isobutylene-co-maleic acid),Poly(methyl vinyl ether-alt-maleic acid), Poly(methyl vinylether-alt-maleic acid monoethyl ester), Poly(methyl vinylether-alt-maleic anhydride), Poly(methyl vinyl ether-alt-maleicanhydride) cross-linked with 1,9-decadiene powder, and/orPoly(styrene-alt-maleic acid) sodium salt.

In one embodiment, the particle is a liposome. In a further embodiment,the particle is a liposome composed of the following lipids at thefollowing molar ratios—30:30:40phosphatidylcholine:phosphatidylglycerol:cholesterol. In yet a furtherembodiment, the particle is encapsulated within a liposome.

It is not necessary that each particle be uniform in size, although theparticles must generally be of a size sufficient to be sequestered inthe spleen or liver and trigger phagocytosis or uptake through receptoror non-receptor mediated mechanism by an antigen presenting cell,including endothelial cell or other MPS cell. Preferably, the particlesare microscopic or nanoscopic in size, in order to enhance solubility,avoid possible complications caused by aggregation in vivo and tofacilitate pinocytosis. Particle size can be a factor for uptake fromthe interstitial space into areas of lymphocyte maturation. A particlehaving a diameter of from about 0.1 μm to about 10 μm is capable oftriggering phagocytosis. Thus in one embodiment, the particle has adiameter within these limits. In another embodiment, the particle has anaverage diameter of about 0.3 μm to about 5 μm. In still anotherembodiment, the particle has an average diameter of about 0.5 μm toabout 3 μm. In another embodiment, the particle has an average diameterof about 0.2 μm to about 2 μm. In a further embodiment the particle hasan average size of about 0.1 μm, or about 0.2 μm or about 0.3 μm orabout 0.4 μm or about 0.5 μm or about 1.0 μm or about 1.5 μm or about2.0 μm or about 2.5 μm or about 3.0 μm or about 3.5 μm or about 4.0 μmor about 4.5 μm or about 5.0 μm. In a particular embodiment the particlehas an average size of about 0.5 μm. In some embodiments, the overallweights of the particles are less than about 10,000 kDa, less than about5,000 kDa, or less than about 1,000 kDa, 500 kDa, 400 kDa, 300 kDa, 200kDa, 100 kDa, 50 kDa, 20 kDa, 10 kDa. The particles in a compositionneed not be of uniform diameter. By way of example, a pharmaceuticalformulation may contain a plurality of particles, some of which areabout 0.5 μm, while others are about 1.0 μm. Any mixture of particlesizes within these given ranges will be useful.

The particles of the current invention can possess a particular zetapotential. In certain embodiments, the zeta potential is negative. Inone embodiment, the zeta potential is less than about −100 mV. In oneembodiment, the zeta potential is less than about −50 mV. In certainembodiments, the particles possess a zeta potential between −100 mV and0 mV. In a further embodiment, the particles possess a zeta potentialbetween −75 mV and 0 mV. In a further embodiment, the particles possessa zeta potential between −60 mV and 0 mV. In a further embodiment, theparticles possess a zeta potential between −50 mV and 0 mV. In still afurther embodiment, the particles possess a zeta potential between −40mV and 0 mV. In a further embodiment, the particles possess a zetapotential between −30 mV and 0 mV. In a further embodiment, theparticles possess a zeta potential between −20 mV and +0 mV. In afurther embodiment, the particles possess a zeta potential between −10mV and −0 mV. In a further embodiment, the particles possess a zetapotential between −100 mV and −50 mV. In another particular embodiment,the particles possess a zeta potential between −75 mV and −50 mV. In aparticular embodiment, the particles possess a zeta potential between−50 mV and −40 mV.

In some embodiments, the charge of a carrier (e.g., positive, negative,neutral) is selected to impart application-specific benefits (e.g.,physiological compatibility, beneficial surface-peptide interactions,etc.). In some embodiments, a carrier has a net neutral or negativecharge (e.g., to reduce non-specific binding to cell surfaces which, ingeneral, bear a net negative charge). In certain embodiments carriersare capable of being conjugated, either directly or indirectly, to anantigen to which tolerance is desired (also referred to herein as anantigen-specific peptide, antigenic peptide, autoantigen, inducingantigen or tolerizing antigen). In some instances, a carrier hasmultiple binding sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 20 . . .50 . . . 100, or more) in order to have multiple copies of anantigen-specific peptide, or multiple different peptides, exposed on thesurface (e.g., to increase the likelihood of a tolerance response). Insome embodiments, a carrier displays a single type of antigenic peptide.In some embodiments, a carrier displays multiple different antigenicpeptides on the surface. In some embodiments, a carrier surface displaysfunctional groups for the covalent attachment of selected moieties(e.g., antigenic peptides). In some embodiments, carrier surfacefunctional groups provide sites for non-covalent interaction withselected moieties (e.g., antigenic peptides). In some embodiments, acarrier has a surface to which conjugating moieties may be adsorbedwithout chemical bond formation.

The size and charge of the particles are critical for toleranceinduction. While the particles will differ in size and charge based onthe antigen encapsulated within them (See Table 1 for examples ofspecific particles), in general, particles of the current invention areeffective at inducing tolerance when they are between about 100nanometers and about 1500 nanometers and have a charge of 0 to about −70mV and are most effective at inducing tolerance when they are 400-800microns and have a charge of between about −25 mV and −70 mV.Furthermore, as shown in Table 1, due in part to the concentration ofthe particles and presence of sucrose and D-mannitol in thelyophilisation process, the average particle size and charge of theparticles can be slightly altered in the lyophilisation process,therefore, both post-synthesis averages and post-lyophilization averagesare given below. As used herein, the term “post-synthesis size” and“post synthesis charge” refer to the size and charge of the particleprior to lyophilization. The term “post lyophilization size” and “postlyophilization charge” refer to the size and charge of the particleafter lyophilization.

TABLE 1 REPRESENTATIVE PARTICLE ANALYSIS Average Average Size postCharge post synthesis synthesis (post (post lyoph- lyoph- ParticleCopolymer Surfactant ilisation) ilisation) Antigen Material ratio usednm mV OVA PLGA 50:50 PEMA 566.5 51.2 (carbox- (538.5) (66.0) ylated)Insulin PLGA 50:50 PEMA 500.9 48.4 (carbox- (385.2) (53.7) ylated)PLP₁₃₉₋₁₅₁ PLGA 50:50 PEMA 429.9 53.7 (carbox- (359.6) (69.4) ylated)Gliaden PLGA 50:50 PEMA 606.1 48.8 (carbox- (1104.0)  (68.1) ylated)Proinsulin PLGA 50:50 PEMA 566.6 44.1 (carbox- (407.2) (49.7) ylated)Lysozyme PLGA 50:50 PEMA 435.3 48.1 (carbox- (393.3) (65.1) ylated) IGRPPLGA 50:50 PEMA 612.5 41.0 (carbox- (399.3) (57.6) ylated) Empty PLGA50:50 PEMA 383.9 52.6 particle (carbox- (343.3) (61.3) (No As) ylated)

In some embodiments, the particle is non-metallic. In these embodimentsthe particle may be formed from a polymer. In a preferred embodiment,the particle is biodegradable in an individual. In this embodiment, theparticles can be provided in an individual across multiple doses withoutthere being an accumulation of particles in the individual. Examples ofsuitable particles include polystyrene particles, PLGA particles,PLURIONICS stabilized polypropylene sulfide particles, and diamondparticles.

Preferably the particle surface is composed of a material that minimizesnon-specific or unwanted biological interactions. Interactions betweenthe particle surface and the interstitium may be a factor that plays arole in lymphatic uptake. The particle surface may be coated with amaterial to prevent or decrease non-specific interactions. Stericstabilization by coating particles with hydrophilic layers such aspoly(ethylene glycol) (PEG) and its copolymers such as PLURONICS®(including copolymers of poly(ethylene glycol)-bl-poly(propyleneglycol)-bl-poly(ethylene glycol)) may reduce the non-specificinteractions with proteins of the interstitium as demonstrated byimproved lymphatic uptake following subcutaneous injections. All ofthese facts point to the significance of the physical properties of theparticles in terms of lymphatic uptake. Biodegradable polymers may beused to make all or some of the polymers and/or particles and/or layers.Biodegradable polymers may undergo degradation, for example, by a resultof functional groups reacting with the water in the solution. The term“degradation” as used herein refers to becoming soluble, either byreduction of molecular weight or by conversion of hydrophobic groups tohydrophilic groups. Polymers with ester groups are generally subject tospontaneous hydrolysis, e.g., polylactides and polyglycolides.

Particles of the present invention may also contain additionalcomponents. For example, carriers may have imaging agents incorporatedor conjugated to the carrier. An example of a carrier nanosphere havingan imaging agent that is currently commercially available is the KodakX-sight nanospheres. Inorganic quantum-confined luminescentnanocrystals, known as quantum dots (QDs), have emerged as ideal donorsin FRET applications: their high quantum yield and tunablesize-dependent Stokes Shifts permit different sizes to emit from blue toinfrared when excited at a single ultraviolet wavelength. (Bruchez, etal., Science, 1998, 281, 2013; Niemeyer, C. M Angew. Chem. Int. Ed.2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, L.E. J. Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybridorganic/inorganic quantum dots based on a class of polymers known asdendrimers, may used in biological labeling, imaging, and opticalbiosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886).Unlike the traditional synthesis of inorganic quantum dots, thesynthesis of these hybrid quantum dot nanoparticles does not requirehigh temperatures or highly toxic, unstable reagents. (Etienne, et al.,Appl. Phys. Lett. 87, 181913, 2005).

Particles can be formed from a wide range of materials. The particle ispreferably composed of a material suitable for biological use. Forexample, particles may be composed of glass, silica, polyesters ofhydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, orcopolymers of hydroxy carboxylic acids and dicarboxylic acids. Moregenerally, the carrier particles may be composed of polyesters ofstraight chain or branched, substituted or unsubstituted, saturated orunsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl,aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxyhydroxy acids, or polyanhydrides of straight chain or branched,substituted or unsubstituted, saturated or unsaturated, linear orcross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl,alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids.Additionally, carrier particles can be quantum dots, or composed ofquantum dots, such as quantum dot polystyrene particles (Joumaa et al.(2006) Langmuir 22: 1810-6). Carrier particles including mixtures ofester and anhydride bonds (e.g., copolymers of glycolic and sebacicacid) may also be employed. For example, carrier particles may comprisematerials including polyglycolic acid polymers (PGA), polylactic acidpolymers (PLA), polysebacic acid polymers (PSA),poly(lactic-co-glycolic) acid copolymers (PLGA or PLG; the terms areinterchangeable), [rho]oly(lactic-co-sebacic) acid copolymers (PLSA),poly(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfidepolymers, poly(caprolactone), chitosan, etc. Other biocompatible,biodegradable polymers useful in the present invention include polymersor copolymers of caprolactones, carbonates, amides, amino acids,orthoesters, acetals, cyanoacrylates and degradable urethanes, as wellas copolymers of these with straight chain or branched, substituted orunsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, oraromatic hydroxy- or di-carboxylic acids. In addition, the biologicallyimportant amino acids with reactive side chain groups, such as lysine,arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine andcysteine, or their enantiomers, may be included in copolymers with anyof the aforementioned materials to provide reactive groups forconjugating to antigen peptides and proteins or conjugating moieties.Biodegradable materials suitable for the present invention includediamond, PLA, PGA, polypropylene sulfide, and PLGA polymers.Biocompatible but non-biodegradable materials may also be used in thecarrier particles of the invention. For example, non-biodegradablepolymers of acrylates, ethylene-vinyl acetates, acyl substitutedcellulose acetates, non-degradable urethanes, styrenes, vinyl chlorides,vinyl fluorides, vinyl imidazoles, chlorosulphonated olefins, ethyleneoxide, vinyl alcohols, TEFLON® (DuPont, Wilmington, Del.), and nylonsmay be employed.

The particles of the instant invention can be manufactured by any meanscommonly known in the art. Exemplary methods of manufacturing particlesinclude, but are not limited to, microemulsion polymerization,interfacial polymerization, precipitation polymerization, emulsionevaporation, emulsion diffusion, solvent displacement, and salting out(Astete and Sabliov, J. Biomater. Sci. Polymer Edn., 17:247-289(2006)).Manipulation of the manufacturing process for PLGA particles can controlparticle properties (e.g. size, size distribution, zeta potential,morphology, hydrophobicity/hydrophilicity, polypeptide entrapment,etc.). The size of the particle is influenced by a number of factorsincluding, but not limited to, the concentration of PLGA, the solventused in the manufacture of the particle, the nature of the organicphase, the surfactants used in manufacturing, the viscosity of thecontinuous and discontinuous phase, the nature of the solvent used, thetemperature of the water used, sonication, evaporation rate, additives,shear stress, sterilization, and the nature of any encapsulated antigenor polypeptide.

Particle size is affected by the polymer concentration; higher particlesare formed from higher polymer concentrations. For example, an increasein PLGA concentration from 1% to 4% (w/v) can increase mean particlesize from about 205 nm to about 290 nm when the solvent propylenecarbonate is used. Alternatively, in ethyl acetate and 5% PluronicF-127, an increase in PLGA concentration from 1% to 5% (w/v) increasesthe mean particle size from 120 nm to 230 nm.

The viscosity of the continuous and discontinuous phase is also animportant parameter that affects the diffusion process, a key step informing smaller particles. The size of the particles increases with anincrease in viscosity of the dispersed phase, whereas the size of theparticles decreases with a more viscous continuous phase. In general,the lower the phase ratio of organic to aqueous solvent, the smaller theparticle size.

Homogenizer speed and agitation also affect particle size; in general,higher speeds and agitation cause a decrease in particle size, althoughthere is a point where further increases in speed and agitation nolonger decrease particle size. There is a favorable impact in the sizereduction when the emulsion is homogenized with a high pressurehomogenizer compared with just high stirring. For example, at a phaseration of 20% in 5% PVA, the mean particle size with stirring is 288 nmand the mean particle size with homogenization (high pressure of 300bars) is 231 nm.

An important size reduction of the particles can be achieved by varyingthe temperature of the water added to improve the diffusion of thesolvent. The mean particle size decreases with an increase in watertemperature.

The nature of the polypeptide encapsulated in the particle also affectsparticle size. In general, encapsulation of hydrophobic polypeptidesleads to the formation of smaller particles compared with theencapsulation of more hydrophilic polypeptides. In the double emulsionprocess, the entrapment of more hydrophilic polypeptides is improved byusing high molecular mass PLGA and a high molecular mass of the firstsurfactant which causes a higher inner phase viscosity. The interactionbetween the solvent, polymer, and polypeptide affects the efficiency ofincorporating the polypeptide into the particle.

The PLGA molecular mass impacts the final mean particle size. Ingeneral, the higher the molecular mass, the higher the mean particlesize. For example, as the composition and molecular mass of PLGA varies(e.g. 12 to 48 kDa for 50:50 PLGA; 12 to 98 kDa for 75:25 PLGA) the meanparticle size varies (about 102 nm −154 nm; about 132 nm to 152 nmrespectively). Even when particles are the same molecular mass, theircomposition can affect average particle size; for example, particleswith a 50:50 ratio generally form particles smaller than those with a75:25 ratio. The end groups on the polymer also affects particle size.For example, particles prepared with ester end-groups form particleswith an average size of 740 nm (PI=0.394) compared with the mean sizefor the acid PLGA end-group is 240 nm (PI=0.225).

The solvent used can also affect particle size; solvents that reduce thesurface tension of the solution also reduce particle size.

The organic solvent is removed by evaporation in a vacuum to avoidpolymer and polypeptide damage and to promote final particle sizereduction. Evaporation of the organic solvent under vacuum is moreefficient in forming smaller particles. For example, evaporation invacuum produces a mean particle size around 30% smaller than the meanparticle size produced under a normal rate of evaporation.

The amplitude of the sonication wavelength also affects the particlecharacteristics. The amplitude of the wavelength should be over 20% with600 to 800 s of sonication to form sable miniemulsions with no moredroplet size changes. However, the main draw-back of sonication is thelack of monodispersity of the emulsion formed.

Organic phases that may be used in the production of the particles ofthe invention include, but are not limited to, ethyl acetate, methylethyl ketone, propylene carbonate, and benzyl alcohol. The continuousphases that may be used, include but are not limited to the surfactantpoloxamer 188.

A variety of surfactants can be used in the manufacturing of theparticles of the invention. The surfactant can be anionic, cationic, ornonionic. Surfactants in the poloxamer and poloaxamines family arecommonly used in particle synthesis. Surfactants that may be used,include, but are not limited to PEG, Tween-80, gelatin, dextran,pluronic L-63, PVA, methylcellulose, lecithin and DMAB. Additionally,biodegradable and biocompatible surfactants including, but not limitedto, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate).In certain embodiments, two surfactants are needed (e.g. in the doubleemulsion evaporation method). These two surfactants can include ahydrophobic surfactant for the first emulsion, and a hydrophobicsurfactant for the second emulsion.

Solvents that may be used in the production of the particles of theinvention include, but are not limited to, acetone, Tetrahydrofuran(THF), chloroform, and members of the chlorinate family, methylchloride. The choice of organic solvents require two selection criteria:the polymer must be soluble in this solvent, and the solvent must becompletely immiscible with the aqueous phase.

Salts that may be used in the production of the particles of theinvention include, but are not limited to magnesium chloridehexahydrate, magnesium acetate tetrahydrate.

Common salting-out agents include, but are not limited to, electrolytes(e.g. sodium chloride, magnesium acetate, magnesium chloride), ornon-electrolytes (e.g. sucrose).

The stability and size of the particles of the invention may be improvedby the addition of compounds including, but not limited to, fatty acidsor short chains of carbons. The addition of the longer carbon chain oflauric acid is associated with the improvement of particlecharacteristics. Furthermore, the addition of hydrophobic additives canimprove the particle size, incorporation of the polypeptide into theparticle, and release profile. Preparations of particles can bestabilized by lyophilization. The addition of a cryoprotectant such astrehalose can decrease aggregation of the particles upon lyophilization.

Suitable beads which are currently available commercially includepolystyrene beads such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

In some embodiments, the present invention provides systems comprising:(a) a delivery scaffold configured for the delivery of chemical and/orbiological agents to a subject; and (b) antigen-coupledpoly(lactide-co-glycolide) particles for induction of antigen-specifictolerance. In some embodiments, at least a portion of said deliveryscaffold is microporous. In some embodiments, the antigen-coupledpoly(lactide-co-glycolide) particles are encapsulated within saidscaffold. In some embodiments, the chemical and/or biological agents areselected from the group consisting of: protein, peptide, smallmolecules, nucleic acids, cells, and particles. In some embodiments,chemical and/or biological agents comprise cell, and said cells comprisepancreatic islet cells.

Physical properties are also related to a nanoparticle's usefulnessafter uptake and retention in areas having immature lymphocytes. Theseinclude mechanical properties such as rigidity or rubberiness. Someembodiments are based on a rubbery core, e.g., a poly(propylene sulfide)(PPS) core with an overlayer, e.g., a hydrophilic overlayer, as in PEG,as in the PPS-PEG system recently developed and characterized forsystemic (but not targeted or immune) delivery. The rubbery core is incontrast to a substantially rigid core as in a polystyrene or metalnanoparticle system. The term rubbery refers to certain resilientmaterials besides natural or synthetic rubbers, with rubbery being aterm familiar to those in the polymer arts. For example, cross-linkedPPS can be used to form a hydrophobic rubbery core. PPS is a polymerthat degrades under oxidative conditions to polysulphoxide and finallypolysulphone, transitioning from a hydrophobic rubber to a hydrophilic,water-soluble polymer. Other sulphide polymers may be adapted for use,with the term sulphide polymer referring to a polymer with a sulphur inthe backbone of the mer. Other rubbery polymers that may be used arepolyesters with glass transition temperature under hydrated conditionsthat is less than about 37° C. A hydrophobic core can be advantageouslyused with a hydrophilic overlayer since the core and overlayer will tendnot to mingle, so that the overlayer tends to sterically expand awayfrom the core. A core refers to a particle that has a layer on it. Alayer refers to a material covering at least a portion of the core. Alayer may be adsorbed or covalently bound. A particle or core may besolid or hollow. Rubbery hydrophobic cores are advantageous over rigidhydrophobic cores, such as crystalline or glassy (as in the case ofpolystyrene) cores, in that higher loadings of hydrophobic drugs can becarried by the particles with the rubbery hydrophobic cores.

Another physical property is the surface's hydrophilicity. A hydrophilicmaterial may have a solubility in water of at least 1 gram per literwhen it is uncrosslinked. Steric stabilization of particles withhydrophilic polymers can improve uptake from the interstitium byreducing non-specific interactions; however, the particles' increasedstealth nature can also reduce internalization by phagocytic cells inareas having immature lymphocytes. The challenge of balancing thesecompeting features has been met, however, and this application documentsthe creation of nanoparticles for effective lymphatic delivery to DCsand other APCs in lymph nodes. Some embodiments include a hydrophiliccomponent, e.g., a layer of hydrophilic material. Examples of suitablehydrophilic materials are one or more of polyalkylene oxides,polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers.The molecular weight of polymers in a layer can be adjusted to provide auseful degree of steric hindrance in vivo, e.g., from about 1,000 toabout 100,000 or even more; artisans will immediately appreciate thatall the ranges and values within the explicitly stated ranges arecontemplated, e.g., between 10,000 and 50,000.

The nanoparticles may incorporate functional groups for furtherreaction. Functional groups for further reaction include electrophilesor nucleophiles; these are convenient for reacting with other molecules.Examples of nucleophiles are primary amines, thiols, and hydroxyls.Examples of electrophiles are succinimidyl esters, aldehydes,isocyanates, and maleimides.

A great variety of means, well known in the art, may be used toconjugate antigenic peptides and proteins to carriers. These methodsinclude any standard chemistries which do not destroy or severely limitthe biological activity of the antigen peptides and proteins, and whichallow for a sufficient number of antigen peptides and proteins to beconjugated to the carrier in an orientation which allows for interactionof the antigen peptide or protein with a cognate T cell receptor.Generally, methods are preferred which conjugate the C-terminal regionsof an antigen peptide or protein, or the C-terminal regions of anantigen peptide or protein fusion protein, to the earner. The exactchemistries will, of course, depend upon the nature of the earnermaterial, the presence or absence of C-terminal fusions to the antigenpeptide or protein, and/or the presence or absence of conjugatingmoieties.

Functional groups can be located on the particle as needed foravailability. One location can be as side groups or termini on the corepolymer or polymers that are layers on a core or polymers otherwisetethered to the particle. For instance, examples are included hereinthat describe PEG stabilizing the nanoparticles that can be readilyfunctionalized for specific cell targeting or protein and peptide drugdelivery.

Conjugates such as ethylene carbodiimide (ECDI), hexamethylenediisocyanate, propyleneglycol di-glycidylether which contain 2 epoxyresidues, and epichlorohydrin may be used for fixation of peptides orproteins to the carrier surface. Without being bound by theory, ECDI issuspected of carrying out two major functions for induction oftolerance: (a) it chemically couples the protein/peptides to the cellsurface via catalysis of peptide bond formation between free amino andfree carboxyl groups; and (b) it induces the carrier to mimic apoptoticcell death such that they are picked up by host antigen presenting cells(which may include endothelial cells) in the spleen and inducetolerance. It is this presentation to host T-cells in a non-immunogenicfashion that leads to direct induction of anergy in autoreactive cells.In addition, ECDI serves as a potent stimulus for the induction ofspecific regulatory T cells.

In one series of embodiments, the antigen peptides and proteins arebound to the carrier via a covalent chemical bond. For example, areactive group or moiety near the C-terminus of the antigen (e.g., theC-terminal carboxyl group, or a hydroxyl, thiol, or amine group from anamino acid side chain) may be conjugated directly to a reactive group ormoiety on the surface of the carrier (e.g., a hydroxyl or carboxyl groupof a PLA or PGA polymer, a terminal amine or carboxyl group of adendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid)by direct chemical reaction. Alternatively, there may be a conjugatingmoiety which covalently conjugates to both the antigen peptides andproteins and the carrier, thereby linking them together.

Reactive carboxyl groups on the surface of a carrier may be joined tofree amines (e.g., from Lys residues) on the antigen peptide or protein,by reacting them with, for example, 1-ethyl-3-[3,9-dimethyl aminopropyl]carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).Similarly, the same chemistry may be used to conjugate free amines onthe surface of a carrier with free carboxyls (e.g., from the C-terminus,or from Asp or Glu residues) on the antigen peptide or protein.Alternatively, free amine groups on the surface of a carrier may becovalently bound to antigen peptides and proteins, or antigen peptide orprotein fusion proteins, using sulfo-SIAB chemistry, essentially asdescribed by Arano et al. (1991) Chem. 2:71-6.

In another embodiment, a non-covalent bond between a ligand bound to theantigen peptide or protein and an anti-ligand attached to the carriermay conjugate the antigen to the carrier. For example, a biotin ligaserecognition sequence tag may be joined to the C-terminus of an antigenpeptide or protein, and this tag may be biotinylated by biotin ligase.The biotin may then serve as a ligand to non-covalently conjugate theantigen peptide or protein to avidin or streptavidin which is adsorbedor otherwise bound to the surface of the carrier as an anti-ligand.Alternatively, if the antigen peptides and proteins are fused to animmunoglobulin domain bearing an Fc region, as described above, the Fcdomain may act as a ligand, and protein A, either covalently ornon-covalently bound to the surface of the carrier, may serve as theanti-ligand to non-covalently conjugate the antigen peptide or proteinto the carrier. Other means are well known in the art which may beemployed to non-covalently conjugate antigen peptides and proteins tocarriers, including metal ion chelation techniques (e.g., using apoly-His tag at the C-terminus of the antigen peptide or protein orantigen peptide or protein fusion proteins, and a Nit-coated carrier),and these methods may be substituted for those described here.

Conjugation of a nucleic acid moiety to a platform molecule can beeffected in any number of ways, typically involving one or morecrosslinking agents and functional groups on the nucleic acid moiety andplatform molecule. Linking groups are added to platforms using standardsynthetic chemistry techniques. Linking groups can be added to nucleicacid moieties using standard synthetic techniques. The practitioner hasa number of choices for antigens used in the combinations of thisinvention. The inducing antigen present in the combination contributesto the specificity of the tolerogenic response that is induced. It mayor may not be the same as the target antigen, which is the antigenpresent or to be placed in the subject being treated which is a targetfor the unwanted immunological response, and for which tolerance isdesired.

An inducing antigen of this invention may be a polypeptide,polynucleotide, carbohydrate, glycolipid, or other molecule isolatedfrom a biological source, or it may be a chemically synthesized smallmolecule, polymer, or derivative of a biological material, providing ithas the ability to induce tolerance according to this description whencombined with the mucosal binding component.

In some embodiments, the present invention provides a carrier (e.g.,immune modifying particle) coupled to one or more peptides,polypeptides, and/or proteins. In some embodiments, a carrier (e.g., PLGcarrier), such as those described herein, are effective to induceantigen-specific tolerance and/or prevent the onset of an immune relateddisease (such as EAE in a mouse model) and/or diminish the severity of apre-existing immune related disease. In some embodiments, thecompositions and methods of the present invention can cause T cells toundertake early events associated with T-cell activation, but do notallow T-cells to acquire effector function. For example, administrationof compositions of the present invention can result in T-cells having aquasi-activated phenotype, such as CD69 and/or CD44 upregulation, but donot display effector function, such as indicated by a lack of IFN-γ orIL-17 synthesis. In some embodiments, administration of compositions ofthe present invention results in T-cells having a quasi-activatedphenotype without having conversion of naïve antigen-specific T-cells toa regulatory phenotype, such as those having CD25^(+/)Foxp3⁺ phenotypes.

In some embodiments, the surface of a carrier (e.g., particle) compriseschemical moieties and/or functional groups that allow attachment (e.g.,covalently, non-covalently) of antigenic peptides and/or otherfunctional elements to the carrier. In some embodiments, the number,orientation, spacing, etc. of chemical moieties and/or functional groupson the carrier (e.g., particle) vary according to carrier chemistry,desired application, etc.

In some embodiments, a carrier comprises one or more biological orchemical agents adhered to, adsorbed on, encapsulated within, and/orcontained throughout the carrier. In some embodiments, a chemical orbiological agent is encapsulated in and/or contained throughout theparticles. The present invention is not limited by the nature of thechemical or biological agents. Such agents include, but are not limitedto, proteins, nucleic acid molecules, small molecule drugs, lipids,carbohydrates, cells, cell components, and the like. In someembodiments, two or more (e.g., 3, 4, 5, etc.) different chemical orbiological agents are included on or within the carrier. In someembodiments, agents are configured for specific release rates. In someembodiments, multiple different agents are configured for differentrelease rates. For example, a first agent may release over a period ofhours while a second agent releases over a longer period of time (e.g.,days, weeks, months, etc.). In some embodiments, the carrier or aportion thereof is configured for slow-release of biological or chemicalagents. In some embodiments, the slow release provides release ofbiologically active amounts of the agent over a period of at least 30days (e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100days, 180 days, etc.). In some embodiments, the carrier or a portionthereof is configured to be sufficiently porous to permit ingrowth ofcells into the pores. The size of the pores may be selected forparticular cell types of interest and/or for the amount of ingrowthdesired. In some embodiments, the particles comprise the antigen ofinterest without other non-peptide active agents, such as drugs orimmunomodulators. Furthermore, in some embodiments the particles of theinvention do not contain immunostimulatory or immunosuppressive peptidesin addition to the antigen of interest. Furthermore, in someembodiments, the particles do not contain other proteins or peptides(e.g. costimulatory molecules, MHC molecules, immunostimulatory peptidesor immunosuppressive peptides) either on the surface or encapsulatedwithin the particle.

Encapsulation of the antigen, biological, and/or chemical agents in theparticle of the invention has been surprisingly found to induceimmunological tolerance and has several advantages. First, theencapsulated particles have a slower cytokine response. Second, whenusing multiple antigens, biological, and/or chemical agents,encapsulation removes the competition between these various moleculesthat might occur if the agents were attached to the surface of theparticle. Third, encapsulation allows more antigens, biological, and/orchemical agents to be incorporated with the particle. Fourth,encapsulation allows for easier use of complex protein antigens or organhomogenates (e.g. pancreas homogenate for type 1 diabetes or peanutextract in peanut allergy). Finally, encapsulation of antigens,biological, and/or chemical agents within the particle instead ofconjugation to the surface of the particle maintains the net negativecharge on the surface of the particle. The encapsulation of the antigen,biological, and/or chemical agents in the particles of the invention maybe performed by any method known in the art. In one embodiment,polypeptide antigens are encapsulated in the particles by adouble-emulsion process. In a further embodiment, the polypeptideantigens are water soluble.

In another embodiment, the polypeptide antigens are encapsulated in theparticles by a single-emulsion process. In a further embodiment, thepolypeptide antigens are more hydrophobic. Sometimes, the doubleemulsion process leads to the formation of large particles which mayresult in the leakage of the hydrophilic active component and lowentrapment efficiencies. The coalescence and Ostwald ripening are twomechanisms that may destabilize the double-emulsion droplet, and thediffusion through the organic phase of the hydrophilic active componentis the main mechanism responsible of low levels of entrapped activecomponent. In some embodiments, it may be beneficial to reduce thenanoparticle size. One strategy to accomplish this is to apply a secondstrong shear rate. The leakage effect can be reduced by using a highpolymer concentration and a high polymer molecular mass, accompanied byan increase in the viscosity of the inner water phase and in increase inthe surfactant molecular mass.

In certain embodiments, the present invention provides carriers havingthereon (or therein) cells or other biological or chemical agents. Wherecells are employed, the carriers are not limited to a particular type ofcells. In some embodiments, the carriers have thereon pancreatic isletcells. In some embodiments, the microporous carriers additionally havethereon ECM proteins and/or exendin-4. The carriers are not limited to aparticular type. In some embodiments, a carrier has regions of varyingporosity (e.g., varying pore size, pore depth, and/or pore density). Insome embodiments, carriers have thereon (or therein) pharmaceuticalagents, DNA, RNA, extracellular matrix proteins, exendin-4, etc. Incertain embodiments, the present invention provides methods fortransplanting pancreatic islet cells with such carriers. In certainembodiments of this invention, the inducing antigen is a single isolatedor recombinantly produced molecule. For treating conditions where thetarget antigen is disseminated to various locations in the host, it isgenerally necessary that the inducing antigen be identical to orimmunologically related to the target antigen. Examples of such antigensare most polynucleotide antigens, and some carbohydrate antigens (suchas blood group antigens).

Any suitable antigens may find use within the scope of the presentinvention. In some embodiments, the inducing antigen contributes to thespecificity of the tolerogenic response that is induced. The inducingantigen may or may not be the same as the target antigen, which is theantigen present or to be placed in the subject being treated which is atarget for the unwanted immunological response, and for which toleranceis desired.

Where the target antigen is preferentially expressed on a particularorgan, cell, or tissue type, the practitioner again has the option ofusing an inducing antigen which is identical with or immunologicallyrelated to the target antigen. However, there is also the additionaloption of using an antigen which is a bystander for the target. This isan antigen which may not be immunologically related to the targetantigen, but is preferentially expressed in a tissue where the targetantigen is expressed. A working theory as to the effectiveness ofbystander suppression is that suppression is an active cell-mediatedprocess that down-regulates the effector arm of the immune response atthe target cells. The suppressor cells are specifically stimulated bythe inducer antigen at the mucosal surface, and home to a tissue sitewhere the bystander antigen is preferentially expressed. Through aninteractive or cytokine-mediated mechanism, the localized suppressorcells then down-regulate effector cells (or inducers of effector cells)in the neighborhood, regardless of what they are reactive against. Ifthe effector cells are specific for a target different from the inducingantigen, then the result is a bystander effect. For further elaborationof the bystander reaction and a list of tolerogenic peptides having thiseffect, the reader is referred to International Patent Publication WO93/16724. An implication of bystander theory is that one of ordinaryskill need not identify or isolate a particular target antigen againstwhich tolerance is desired in order to practice the present invention.The practitioner need only be able to obtain at least one moleculepreferentially expressed at the target site for use as an inducingantigen.

In certain embodiments of this invention, the inducing antigen is not inthe same form as expressed in the individual being treated, but is afragment or derivative thereof. Inducing antigens of this inventioninclude peptides based on a molecule of the appropriate specificity butadapted by fragmentation, residue substitution, labeling, conjugation,and/or fusion with peptides having other functional properties. Theadaptation may be performed for any desirable purposes, including butnot limited to the elimination of any undesirable property, such astoxicity or immunogenicity; or to enhance any desirable property, suchas mucosal binding, mucosal penetration, or stimulation of thetolerogenic arm of the immune response. Terms such as insulin peptide,collagen peptide, and myelin basic protein peptide, as used herein,refer not only to the intact subunit, but also to allotypic andsynthetic variants, fragments, fusion peptides, conjugates, and otherderivatives that contain a region that is homologous (preferably 70%identical, more preferably 80% identical and even more preferably 90%identical at the amino acid level) to at least 10 and preferably 20consecutive amino acids of the respective molecule for which it is ananalog, wherein the homologous region of the derivative shares with therespective parent molecule an ability to induce tolerance to the targetantigen.

It is recognized that tolerogenic regions of an inducing antigen areoften different from immunodominant epitopes for the stimulation of anantibody response. Tolerogenic regions are generally regions that can bepresented in particular cellular interactions involving T cells.Tolerogenic regions may be present and capable of inducing toleranceupon presentation of the intact antigen. Some antigens contain cryptictolerogenic regions, in that the processing and presentation of thenative antigen does not normally trigger tolerance. An elaboration ofcryptic antigens and their identification is found in InternationalPatent Publication WO 94/27634.

In certain embodiments of this invention, two, three, or a higherplurality of inducing antigens is used. It may be desirable to implementthese embodiments when there are a plurality of target antigens, or toprovide a plurality of bystanders for the target. For example, bothinsulin and glucagon can be mixed with a mucosal binding component inthe treatment of diabetes. It may also be desirable to provide acocktail of antigens to cover several possible alternative targets. Forexample, a cocktail of histocompatibility antigen fragments could beused to tolerize a subject in anticipation of future transplantationwith an allograft of unknown phenotype. Allovariant regions of humanleukocyte antigens are known in the art: e.g., Immunogenetics 29:231,1989. In another example, a mixture of allergens may serve as inducingantigen for the treatment of atopy.

Inducing antigens can be prepared by a number of techniques known in theart, depending on the nature of the molecule. Polynucleotide,polypeptide, and carbohydrate antigens can be isolated from cells of thespecies to be treated in which they are enriched. Short peptides areconveniently prepared by amino acid synthesis. Longer proteins of knownsequence can be prepared by synthesizing an encoding sequence orPCR-amplifying an encoding sequence from a natural source or vector, andthen expressing the encoding sequence in a suitable bacterial oreukaryotic host cell.

In certain embodiments of this invention, the combination comprises acomplex mixture of antigens obtained from a cell or tissue, one or moreof which plays the role of inducing antigen. The antigens may be in theform of whole cells, either intact or treated with a fixative such asformaldehyde, glutaraldehyde, or alcohol. The antigens may be in theform of a cell lysate, created by detergent solubilization or mechanicalrupture of cells or tissue, followed by clarification. The antigens mayalso be obtained by subcellular fractionation, particularly anenrichment of plasma membrane by techniques such as differentialcentrifugation, optionally followed by detergent solubilization anddialysis. Other separation techniques are also suitable, such asaffinity or ion exchange chromatography of solubilized membraneproteins.

In one embodiment, the antigenic peptide or protein is an autoantigen,an alloantigen or a transplantation antigen. In yet another particularembodiment, the autoantigen is selected from the group consisting ofmyelin basic protein, collagen or fragments thereof, DNA, nuclear andnucleolar proteins, mitochondrial proteins and pancreatic (3-cellproteins.

The invention provides for the induction of tolerance to an autoantigenfor the treatment of autoimmune diseases by administering the antigenfor which tolerance is desired. For example, autoantibodies directedagainst the myelin basic protein (MBP) are observed in patients withmultiple sclerosis, and, accordingly, MBP antigenic peptides or proteinsmay be used in the invention to be delivered using the compositions ofthe present invention to treat and prevent multiple sclerosis.

By way of another non-limiting example, an individual who is a candidatefor a transplant from a non-identical twin may suffer from rejection ofthe engrafted cells, tissues or organs, as the engrafted antigens areforeign to the recipient. Prior tolerance of the recipient individual tothe intended graft abrogates or reduces later rejection. Reduction orelimination of chronic anti-rejection therapies may be achieved by thepractice of the present invention. In another example, many autoimmunediseases are characterized by a cellular immune response to anendogenous or self antigen. Tolerance of the immune system to theendogenous antigen is desirable to control the disease.

In a further example, sensitization of an individual to an industrialpollutant or chemical, such as may be encountered on-the-job, presents ahazard of an immune response. Prior tolerance of the individual's immunesystem to the chemical, in particular in the form of the chemicalreacted with the individual's endogenous proteins, may be desirable toprevent the later occupational development of an immune response.

Allergens are other antigens for which tolerance of the immune responsethereto is also desirable. In one embodiment, the antigen is a gliaden.In a further embodiment, the antigen is A-gliaden.

Notably, even in diseases where the pathogenic autoantigen is unknown,bystander suppression may be induced using antigens present in theanatomical vicinity. For example, autoantibodies to collagen areobserved in rheumatoid arthritis and, accordingly, a collagen-encodinggene may be utilized as the antigen-expressing gene module in order totreat rheumatoid arthritis (see e.g. Choy (2000) Curr Opin InvestigDrugs 1: 58-62). Furthermore, tolerance to beta cell autoantigens may beutilized to prevent development of type 1 diabetes (see e.g. Bach andChatenoud (2001) Ann Rev Immunol 19: 131-161).

As another example, auto-antibodies directed against myelinoligodendrocyte glycoprotein (MOG) are observed in autoimmuneencephalomyelitis and in many other CNS diseases as well as multiplesclerosis (see e.g. Iglesias et al. (2001) Glia 36: 22-34). Accordingly,use of MOG antigen expressing constructs in the invention allows fortreatment of multiple sclerosis as well as related autoimmune disordersof the central nervous system.

Still other examples of candidate autoantigens for use in treatingautoimmune disease include: pancreatic beta-cell antigens, insulin andGAD to treat insulin-dependent diabetes mellitus; collagen type 11,human cartilage gp 39 (HCgp39) and gp130-RAPS for use in treatingrheumatoid arthritis; myelin basic protein (MBP), proteo lipid protein(PLP) and myelin oligodendrocyte glycoprotein (MOG, see above) to treatmultiple sclerosis; fibrillarin, and small nucleolar protein (snoRNP) totreat scleroderma; thyroid stimulating factor receptor (TSH-R) for usein treating Graves' disease; nuclear antigens, histones, glycoproteingp70 and ribosomal proteins for use in treating systemic lupuserythematosus; pyruvate dehydrogenase dehydrolipoamide acetyltransferase(PCD-E2) for use in treating primary billiary cirrhosis; hair follicleantigens for use in treating alopecia areata; and human tropomyosinisoform 5 (hTM5) for use in treating ulcerative colitis.

In one embodiment, the particles of the invention are coupled toantigens comprising one or more epitopes associated with allergies,autoimmune diseases and/or inflammatory diseases or disorders. Theantigens may comprise one or more copies of an epitope. In oneembodiment, the antigens comprise a single epitope associated with onedisease or disorder. In a further embodiment, the antigens comprise morethan one epitope associated with the same disease or disorder. In yet afurther embodiment, the antigens comprise more than one epitopeassociated with different diseases or disorders. In a furtherembodiment, the antigens comprise one or more epitopes associated withone or more allergies. In a further embodiment, the antigens compriseone or more epitopes associated with multiple sclerosis, type 1diabetes. Celiac's disease, and/or inflammatory bowel disease, includingCrohn's disease or ulcerative colitis. In one embodiment, the epitopesare from myelin basic protein (e.g. SEQ ID NOs:4975 & 4976), proteolipidprotein (e.g. SEQ ID NO: 4977), myelin oligodendrocyte glycoprotein(e.g. SEQ ID NOs: 1 & 4978), aquaporin, (e.g. SEQ ID NO: 4979), myelinassociated glycoprotein (e.g. SEQ ID NO: 4980), insulin (e.g. SEQ ID NO:4981), glutamic acid decarboxylase (e.g. SEQ ID NO: 4982), gliadin (e.g.SEQ ID NOs:4983-4985 or 5136-5140), the α3 chain of type IV collagen(e.g. SEQ ID NO: 5017), or fragments, homologs, or isoforms thereof. Ina further embodiment, the epitopes are from gluten, including fromgliadin and/or glutenin. In one embodiment, the epitopes are frominsulin homologs, such as those described in U.S. Pat. No. 8,476,228hereby incorporated in its entirety for all purposes. In one embodiment,the gliaden epitopes are SEQ ID NOs: 13, 14, 16, 320, or 321 in U.S.Application No. 20110293644, hereby incorporated in its entirety for allpurposes.

Further non-limiting examples of epitopes associated with variousautoimmune diseases and/or inflammatory diseases or disorders that arecontemplated by the instant invention are described in Tables 2 and 3.

TABLE 2 Representative Linear Epitopes Disease Representative EpitopesMultiple Sclerosis SEQ ID NOs: 2-1294 Celiac SEQ ID NOs: 1295-1724;Disease SEQ ID NOs: 1726-1766; SEQ ID NOs: 4986-5140 Diabetes SEQ IDNOs: 1767-1840; SEQ ID NOs: 1842-1962; SEQ ID NOs: 1964-2027; SEQ IDNOs: 2029-2073; SEQ ID NOs: 2075-2113; SEQ ID NOs: 2115-2197; SEQ IDNOs: 2199-2248; SEQ ID NOs: 2250-2259; SEQ ID NOs: 2261-2420; SEQ IDNOs: 2422-2486; SEQ ID NOs: 2489-2505 Rheumatoid SEQ ID NOs: 2506-3260;Arthritis SEQ ID NOs: 3262-3693 Systemic SEQ ID NOs: 3694-3857; LupusSEQ ID NOs: 3860-4565 Erythematosus Good Pasture's SEQ ID NOs:4566-4576; Syndrome SEQ ID NOs: 4578-4610; SEQ ID NOs: 4612-4613; SEQ IDNOs: 5018-5039 Autoimmune SEQ ID NOs: 4614-4653 Uveitis Autoimmune SEQID NOs: 4654-4694; Thyroiditis SEQ ID NOs: 4696-4894; SEQ ID NOs:4896-4901 Autoimmune Myositis SEQ ID NOs: 4902-4906 AutoimmuneVasculitis SEQ ID NOs: 4907-4914 Autoimmune Pancreatitis SEQ ID NOs:4915-4917 Crohns Disease SEQ ID NOs: 4918-4941 Ulcerative Colitis SEQ IDNOs: 4942-4952 Psoriasis SEQ ID NOs: 4953-4963 Reactive Arthritis SEQ IDNOs: 4964-4974

Not all epitopes are linear epitopes; epitopes can also bediscontinuous, conformational epitopes. A number of discontinuousepitopes associated with autoimmune diseases or inflammatory diseasesand/or disorders are known. Non-limiting examples of discontinuousepitopes are described in Table 3.

TABLE 3 Representative Discontinuous Epitopes Disease Epitope FullLength Polypeptide Celiac D151, E153, E154, E155, Protein-glutamineDisease E158; gamma-gluta- D306, N308, N310; myltransferase 2 D434,E435, E437, D438; SEQ ID NO: 1725 E329; E153; R19, E153, M659; or C277,H335, D358 Diabetes R255, F256, K257, K263, Glutamate decarbox- E264,K265, L270, P271, ylase 2 R272, L273, L285, K286, SEQ ID NOs: 1841,K287, I294, G295, T296, 1963, 2114, & 2249 D297, S298, R317, R318; N483,I484, I485, K486, N487, R488, E489, G490, Y491, E492, M493, V494, F495,D496, G497, K498, P499, F556, F557, R558, M559, V560, I561, S562, N563,P564, A565, A566, T567, H568, Q569, D570, I571, D572, F573, L574, I575,E576, E577, I578, E579, R580, L581, G582, Q583, D584, L585; E264; E517,E520, E521, S524, S527, V532; E517, E521; K358; R536, Y540 DiabetesP876, A877, E878, T880; protein tyrosine T804; phosphatase, receptorT804, V813, D821, R822, type, N precursor Q862, P886; SEQ ID NOs: 2028 &T804, V813, D821, R822, 2074 Q862, P886; W799, E836, N858; D911; Q862;L831, H833, V834, E836, Q860; W799, E836, N858; W799, L831, H833, V834,Y835, E836, Q860; Diabetes R325, R332, E333, K336, zinc transporter 8K340; isoform a R325; SEQ ID NO: 2421 W325 Diabetes E872, C945Receptor-type tyrosine-protein phosphatase N2 SEQ ID NOs: 2198, 2260, &2487 Diabetes W799, C909 tyrosine phosphatase SEQ ID NO: 2488 RheumatoidL14, M15, I16, S17, R18, Chain A, Crystal Arthritis N147, G148, S187,M191, Structure Of A H196, N197, H198, Y199, Human Igm Rheumatoid Q201,S203 Factor Fab In Complex With Its Autoantigen Igg Fc SEQ ID NO: 3261Systemic K591, S592, G593 ATP-dependent Lupus DNA helicase 2Erythematosus subunit 1 SEQ ID NO: 3858 Systemic M1, K2, L3, V4, R5, F6,L7, Small nuclear Lupus M8, K9, L10, S11, H12, ribonucleoproteinErythematosus E13, T14, V15, T16, I17, Sm D1 E18, L19, K20, N21, G22,SEQ ID NO: 3859 T23, Q24, V25, H26, P85, K86, V87, K88, S89, K90, K91,R92, E93, A94, V95, A96, G97, R98, G99, R100, G101, R102, G103, R104,G105, R106, G107, R108, G109, R110, G111, R112, G113, R114, G115, G116,P117, R118, R119 Systemic G59, R62 beta-2- Lupus glycoprotein IErythematosus SEQ ID NO: 4357 Good T24, A25, I26, S28, E31, type IVcollagen Pasture's V34, P35, S38, Q64 alpha3 chain Syndrome SEQ ID NO:4577 Good T1455, A1456, I1457, alpha3 type IV Pasture's S1459, E1462,T1464, collagen Syndrome V1465, P1466, Y1468, SEQ ID NO: 4611 S1469,Q1495, T1537, T1565, P1569, H1572, K1579, A1634 Autoimmune E604, D620,K627, D630; Thyroid peroxidase Thyroiditis R225, R646, D707; SEQ ID NO:4695 K627; R225; Y772; K713, F714, P715, E716; P715, D717 AutoimmuneD36, R38, K42, Q55, K58, Thyrotropin receptor Thyroiditis I60, E61, R80,Y82, S84, SEQ ID NO: 4895 T104, H105, E107, R109, N110, K129, F130,D151, F153, I155, E157, T181, K183, D203

Combinations of antigens and/or epitopes can be tested for their abilityto promote tolerance by conducting experiments with isolated cells or inanimal models.

In some embodiments, the tolerance inducing compositions of the presentinvention contain an apoptosis signaling molecule (e.g., in addition toan antigenic peptide or other antigenic molecule). In some embodiments,the apoptosis signaling molecule is coupled and/or associated with thesurface of the carrier. In some embodiments an apoptotic signalingmolecules allows a carrier to be perceived as an apoptotic body byantigen presenting cells of the host, such as cells of the hostreticuloendothelial system; this allows presentation of the associatedpeptide epitopes in a tolerance-inducing manner. Without being bound bytheory, this is presumed to prevent the upregulation of moleculesinvolved in immune cell stimulation, such as MHC class I/II, andcostimulatory molecules. These apoptosis signaling molecules may alsoserve as phagocytic markers. For example, apoptosis signaling moleculessuitable for the present invention have been described in US Pat App No.20050113297, which is hereby incorporated by reference in its entirety.Molecules suitable for the present invention include molecules thattarget phagocytes, which include macrophages, dendritic cells,monocytes, granulocytes and neutrophils.

In some embodiments, molecules suitable as apoptotic signallingmolecules act to enhance tolerance of the associated peptides.Additionally, a carrier bound to an apoptotic signaling molecule can bebound by C1q in apoptotic cell recognition (Paidassi et al., (2008) J.Immunol. 180:2329-2338; herein incorporated by reference in itsentirety). For example, molecules that may be useful as apoptoticsignalling molecules include phosphatidyl serine, annexin-1, annexin-5,milk fat globule-EGF-factor 8 (MFG-E8), or the family of thrombospondins(e.g., thrombospondin-1 (TSP-1)). Various molecules suitable for use asapoptotic signalling molecules with the present invention are discussed,for example, in U.S. Pat. App. 2012/0076831; herein incorporated byreference in its entirety).

In some embodiments, the apoptotic signalling molecule may be conjugatedto the antigen-specific peptide. In some instances, the apoptoticsignalling molecule and antigen-specific peptide are conjugated by thecreation of a fusion protein. For example a fusion protein may compriseat least one antigen-specific peptide (or a fragment or a variantthereof) coupled to at least one molecule of an apoptotic signallingmolecule (or a fragment or a variant thereof). For the creation offusion proteins, the terms “fusion protein,” “fusion peptide,” “fusionpolypeptide,” and “chimeric peptide” are used interchangeably. Suitablefragments of the antigen-specific peptide include any fragment of thefull-length peptide that retains the function of generating the desiredantigen-specific tolerance function of the present invention. The fusionprotein may be created by various means understood in the art (e.g.,genetic fusion, chemical conjugation, etc.). The two proteins may befused either directly or via an amino acid linker. The polypeptidesforming the fusion protein are typically linked C-terminus toN-terminus, although they can also be linked C-terminus to C-terminus,N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptidesof the fusion protein can be in any order. A peptide linker sequence maybe employed to separate the first and second polypeptide components by adistance sufficient to ensure that each polypeptide folds into itssecondary and tertiary structures. Amino acid sequences which may beusefully employed as linkers include those disclosed in Maratea et. al.,Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. Nos. 4,935,233 and 4,751,180; hereinincorporated by reference in their entireties. The linker sequence maygenerally be from 1 to about 50 amino acids in length. In someembodiments, linker sequences are not required and/or utilized, forexample, when the first and second polypeptides have non-essentialN-terminal amino acid regions that can be used to separate thefunctional domains and prevent steric interference.

A proxy for tolerogenic activity is the ability of an intact antigen orfragment to stimulate the production of an appropriate cytokine at thetarget site. The immunoregulatory cytokine released by T suppressorcells at the target site is thought to be TGF-β (Miller et al., Proc.Natl. Acad. Sci. USA 89:421, 1992). Other factors that may be producedduring tolerance are the cytokines IL4 and IL-10, and the mediator PGE.In contrast, lymphocytes in tissues undergoing active immune destructionsecrete cytokines such as IL-I, IL-2, IL-6, and γ-IFN. Hence, theefficacy of a candidate inducing antigen can be evaluated by measuringits ability to stimulate the appropriate type of cytokines.

With this in mind, a rapid screening test for tolerogenic epitopes ofthe inducing antigen, effective mucosal binding components, effectivecombinations, or effective modes and schedules of mucosal administrationcan be conducted using syngeneic animals as donors for in vitro cellassays. Animals are treated at a mucosal surface with the testcomposition, and at some time are challenged with parenteraladministration of the target antigen in complete Freund's adjuvant.Spleen cells are isolated, and cultured in vitro in the presence of thetarget antigen at a concentration of about 50 μg/mL. Target antigen canbe substituted with candidate proteins or sub-fragments to map thelocation of tolerogenic epitopes. Cytokine secretion into the medium canbe quantitated by standard immunoassay.

The ability of the cells to suppress the activity of other cells can bedetermined using cells isolated from an animal immunized with the targetantigen, or by creating a cell line responsive to the target antigen(Ben-Nun et al., Eur. J. Immunol. 11:195, 1981, herein incorporated byreference in its entirety). In one variation of this experiment, thesuppressor cell population is mildly irradiated (about 1000 to 1250rads) to prevent proliferation, the suppressors are co-cultured with theresponder cells, and then tritiated thymidine incorporation (or MTT) isused to quantitate the proliferative activity of the responders. Inanother variation, the suppressor cell population and the responder cellpopulation are cultured in the upper and lower levels of a dual chambertranswell culture system (Costar, Cambridge Mass.), which permits thepopulations to coincubate within 1 mm of each other, separated by apolycarbonate membrane (WO 93/16724). In this approach, irradiation ofthe suppressor cell population is unnecessary, since the proliferativeactivity of the responders can be measured separately.

In embodiments of the invention where the target antigen is alreadypresent in the individual, there is no need to isolate the antigen orprecombine it with the mucosal binding component. For example, theantigen may be expressed in the individual in a certain fashion as aresult of a pathological condition (such as inflammatory bowel diseaseor Celiac disease) or through digestion of a food allergen. Testing isperformed by giving the mucosal binding component in one or more dosesor formulations, and determining its ability to promote tolerizationagainst the antigen in situ.

The effectiveness of compositions and modes of administration fortreatment of specific disease can also be elaborated in a correspondinganimal disease model. The ability of the treatment to diminish or delaythe symptomatology of the disease is monitored at the level ofcirculating biochemical and immunological hallmarks of the disease,immunohistology of the affected tissue, and gross clinical features asappropriate for the model being employed. Non-limiting examples ofanimal models that can be used for testing are included in the followingsection.

The invention contemplates modulation of tolerance by modulating TH1response, TH2 response, TH17 response, or a combination of theseresponses. Modulating TH1 response encompasses changing expression of,e.g., interferon-gamma. Modulating TH2 response encompasses changingexpression of, e.g., any combination of IL-4, IL-5, IL-10, and IL-13.Typically an increase (decrease) in TH2 response will comprise anincrease (decrease) in expression of at least one of IL-4, IL-5, IL-10,or IL-13; more typically an increase (decrease) in TH2 response willcomprise an increase in expression of at least two of IL-4, IL-5, IL-10,or EL-13, most typically an increase (decrease) in TH2 response willcomprise an increase in at least three of DL-4, IL-5, IL-10, or IL-13,while ideally an increase (decrease) in TH2 response will comprise anincrease (decrease) in expression of all of IL-4, IL-5, IL-10, andIL-13. Modulating TH 17 encompasses changing expression of, e.g.,TGF-beta, IL-6, IL-21 and IL23, and effects levels of IL-17, IL-21 andIL-22.

Other suitable methods for assessing the effectiveness of compositionsand methods of the present invention are understood in the art, as arediscussed, for example, in U.S. Pat. App. 2012/0076831 (hereinincorporated by reference in its entirety).

Certain embodiments of this invention relate to priming of immunetolerance in an individual not previously tolerized by therapeuticintervention. These embodiments generally involve a plurality ofadministrations of a combination of antigen and mucosal bindingcomponent. Typically, at least three administrations, frequently atleast four administrations, and sometimes at least six administrationsare performed during priming in order to achieve a long-lasting result,although the subject may show manifestations of tolerance early in thecourse of treatment. Most often, each dose is given as a bolusadministration, but sustained formulations capable of mucosal releaseare also suitable. Where multiple administrations are performed, thetime between administrations is generally between 1 day and 3 weeks, andtypically between about 3 days and 2 weeks. Generally, the same antigenand mucosal binding component are present at the same concentration, andthe administration is given to the same mucosal surface, but variationsof any of these variables during a course of treatment may beaccommodated.

Other embodiments of this invention relate to boosting or extending thepersistence of a previously established immune tolerance. Theseembodiments generally involve one administration or a short course oftreatment at a time when the established tolerance is declining or atrisk of declining. Boosting is generally performed 1 month to 1 year,and typically 2 to 6 months after priming or a previous boost. Thisinvention also includes embodiments that involve regular maintenance oftolerance on a schedule of administrations that occur semiweekly,weekly, biweekly, or on any other regular schedule.

The particles of the current invention can be given in any doseeffective to dampen the inflammatory immune response in a subject inneed thereof or to treat a bacterial or viral infection in a subject inneed thereof. In certain embodiments, about 10² to about 10²⁰ particlesare provided to the individual. In a further embodiment between about10³ to about 10¹⁵ particles are provided. In yet a further embodimentbetween about 10⁶ to about 10¹² particles are provided. In still afurther embodiment between about 10⁸ to about 10¹⁰ particles areprovided. In a preferred embodiment the preferred dose is 0.1%solids/ml. Therefore, for 0.5 μm beads, a preferred dose isapproximately 4×10⁹ beads, for 0.05 μm beads, a preferred dose isapproximately 4×10¹² beads, for 3 μm beads, a preferred dose is 2×10⁷beads. However, any dose that is effective in treating the particularcondition to be treated is encompassed by the current invention.

The invention is useful for treatment of immune related disorders suchas autoimmune disease, transplant rejection, enzyme deficiencies andallergic reactions. Substitution of a synthetic, biocompatible particlesystem to induce immune tolerance could lead to ease of manufacturing,broad availability of therapeutic agents, increase uniformity betweensamples, increase the number of potential treatment sites anddramatically reduce the potential for allergic responses to a carriercell.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages. Immune cells involved in the immuneresponse include lymphocytes, such as B cells and T cells (CD4⁺, CD8⁺,Th1 and Th2 cells); antigen presenting cells (e.g., professional antigenpresenting cells such as dendritic cells, macrophages, B lymphocytes,Langerhans cells, and nonprofessional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes. Insome embodiments, the modified particles of the present invention areeffective to reduce inflammatory cell trafficking to the site ofinflammation.

As used herein, the term “anergy,” “tolerance,” or “antigen-specifictolerance” refers to insensitivity of T cells to T cellreceptor-mediated stimulation. Such insensitivity is generallyantigen-specific and persists after exposure to the antigenic peptidehas ceased. For example, anergy in T cells is characterized by lack ofcytokine production, e.g., IL-2. T-cell anergy occurs when T cells areexposed to antigen and receive a first signal (a T cell receptor or CD-3mediated signal) in the absence of a second signal (a costimulatorysignal). Under these conditions, re-exposure of the cells to the sameantigen (even if re-exposure occurs in the presence of a costimulatorymolecule) results in failure to produce cytokines and subsequentlyfailure to proliferate. Thus, a failure to produce cytokines preventsproliferation. Anergic T cells can, however, proliferate if culturedwith cytokines (e.g., IL-2). For example, T cell anergy can also beobserved by the lack of IL-2 production by T lymphocytes as measured byELISA or by a proliferation assay using an indicator cell line.Alternatively, a reporter gene construct can be used. For example,anergic T cells fail to initiate DL-2 gene transcription induced by aheterologous promoter under the control of the 5′ IL-2 gene enhancer orby a multimer of the API sequence that can be found within the enhancer(Kang et al. 1992 Science. 257:1134).

As used herein, the term “immunological tolerance” refers to methodsperformed on a proportion of treated subjects in comparison withuntreated subjects where: a) a decreased level of a specificimmunological response (thought to be mediated at least in part byantigen-specific effector T lymphocytes, B lymphocytes, antibody, ortheir equivalents); b) a delay in the onset or progression of a specificimmunological response; or c) a reduced risk of the onset or progressionof a specific immunological response. “Specific” immunological toleranceoccurs when immunological tolerance is preferentially invoked againstcertain antigens in comparison with others. “Non-Specific” immunologicaltolerance occurs when immunological tolerance is invokedindiscriminately against antigens which lead to an inflammatory immuneresponse. “Quasi-Specific” immunological tolerance occurs whenimmunological tolerance is invoked semi-discriminately against antigenswhich lead to a pathogenic immune response but not to others which leadto a protective immune response.

Tolerance to autoantigens and autoimmune disease is achieved by avariety of mechanisms including negative selection of self-reactive Tcells in the thymus and mechanisms of peripheral tolerance for thoseautoreactive T cells that escape thymic deletion and are found in theperiphery. Examples of mechanisms that provide peripheral T celltolerance include “ignorance” of self antigens, anergy orunresponsiveness to autoantigen, cytokine immune deviation, andactivation-induced cell death of self-reactive T cells. In addition,regulatory T cells have been shown to be involved in mediatingperipheral tolerance. See, for example, Walker et al. (2002) Nat. Rev.Immunol. 2: 11-19; Shevach et al. (2001) Immunol. Rev. 182:58-67. Insome situations, peripheral tolerance to an autoantigen is lost (orbroken) and an autoimmune response ensues. For example, in an animalmodel for EAE, activation of antigen presenting cells (APCs) through TLRinnate immune receptors was shown to break self-tolerance and result inthe induction of EAE (Waldner et al. (2004) J. Clin. Invest.113:990-997).

Accordingly, in some embodiments, the invention provides methods forincreasing antigen presentation while suppressing or reducing TLR7/8,TLR9, and/or TLR 7/8/9 dependent cell stimulation. As described herein,administration of particular modified particles results in antigenpresentation by DCs or APCs while suppressing the TLR 7/8, TLR9, and/orTLR7/8/9 dependent cell responses associated with immunostimulatorypolynucleotides. Such suppression may include decreased levels of one ormore TLR-associated cytokines.

As discussed above this invention provides novel compounds that havebiological properties useful for the treatment of Mac-1 and LFA-1mediated disorders.

Accordingly, in another aspect of the present invention, pharmaceuticalcompositions are provided, which comprise the immune modifying particlesand optionally comprise a pharmaceutically acceptable carrier. Incertain embodiments, these compositions optionally further comprise oneor more additional therapeutic agents. Alternatively, the modifiedparticles of the current invention may be administered to a patient inneed thereof in combination with the administration of one or more othertherapeutic agents. For example, additional therapeutic agents forconjoint administration or inclusion in a pharmaceutical compositionwith a compound of this invention may be an approved anti-inflammatoryagent, or it may be any one of a number of agents undergoing approval inthe Food and Drug Administration that ultimately obtain approval for thetreatment of any disorder characterized by an uncontrolled inflammatoryimmune response or a bacterial or viral infection. It will also beappreciated that certain of the modified particles of present inventioncan exist in free form for treatment, or where appropriate, as apharmaceutically acceptable derivative thereof.

The pharmaceutical compositions of the present invention additionallycomprise a pharmaceutically acceptable carrier, which, as used herein,includes any and all solvents, diluents, or other liquid vehicle,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

The particles of the invention may be administered orally, nasally,intravenously, intramuscularly, ocularly, transdermally,intraperitoneally, or subcutaneously. In one embodiment, the particlesof the invention are administered intravenously.

The effective amounts and method of administration of the presentinvention for modulation of an immune response can vary based on theindividual, what condition is to be treated and other factors evident toone skilled in the art. Factors to be considered include route ofadministration and the number of doses to be administered. Such factorsare known in the art and it is well within the skill of those in the artto make such determinations without undue experimentation. A suitabledosage range is one that provides the desired regulation of immune.Useful dosage ranges of the carrier, given in amounts of carrierdelivered, may be, for example, from about any of the following: 0.5 to10 mg/kg, 1 to 9 mg/kg, 2 to 8 mg/kg, 3 to 7 mg/kg, 4 to 6 mg/kg, 5mg/kg, 1 to 10 mg/kg, 5 to 10 mg/kg. Alternatively, the dosage can beadministered based on the number of particles. For example, usefuldosages of the carrier, given in amounts of carrier delivered, may be,for example, about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or greater number ofparticles per dose. The absolute amount given to each patient depends onpharmacological properties such as bioavailability, clearance rate androute of administration. Details of pharmaceutically acceptablecarriers, diluents and excipients and methods of preparingpharmaceutical compositions and formulations are provided in RemmingtonsPharmaceutical Sciences 18^(th) Edition, 1990, Mack Publishing Co.,Easton, Pa., USA., which is hereby incorporated by reference in itsentirety.

The effective amount and method of administration of the particularcarrier formulation can vary based on the individual patient, desiredresult and/or type of disorder, the stage of the disease and otherfactors evident to one skilled in the art. The route(s) ofadministration useful in a particular application are apparent to one ofskill in the art. Routes of administration include but are not limitedto topical, dermal, transdermal, transmucosal, epidermal, parenteral,gastrointestinal, and naso-pharyngeal and pulmonary, includingtransbronchial and transalveolar. A suitable dosage range is one thatprovides sufficient IRP-containing composition to attain a tissueconcentration of about 1-50 μM as measured by blood levels. The absoluteamount given to each patient depends on pharmacological properties suchas bioavailability, clearance rate and route of administration.

The present invention provides carrier formulations suitable for topicalapplication including, but not limited to, physiologically acceptableimplants, ointments, creams, rinses and gels. Exemplary routes of dermaladministration are those which are least invasive such as transdermaltransmission, epidermal administration and subcutaneous injection.

Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the carrier to penetrate the skinand enter the blood stream. Compositions suitable for transdermaladministration include, but are not limited to, pharmaceuticallyacceptable suspensions, oils, creams and ointments applied directly tothe skin or incorporated into a protective carrier such as a transdermaldevice (so-called “patch”). Examples of suitable creams, ointments etc.can be found, for instance, in the Physician's Desk Reference.Transdermal transmission may also be accomplished by iontophoresis, forexample using commercially available patches which deliver their productcontinuously through unbroken skin for periods of several days or more.Use of this method allows for controlled transmission of pharmaceuticalcompositions in relatively great concentrations, permits infusion ofcombination drugs and allows for contemporaneous use of an absorptionpromoter.

Parenteral routes of administration include but are not limited toelectrical (iontophoresis) or direct injection such as direct injectioninto a central venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Formulations of carrier suitablefor parenteral administration are generally formulated in USP water orwater for injection and may further comprise pH buffers, salts bulkingagents, preservatives, and other pharmaceutically acceptable excipients.Immunoregulatory polynucleotide for parenteral injection may beformulated in pharmaceutically acceptable sterile isotonic solutionssuch as saline and phosphate buffered saline for injection.

Gastrointestinal routes of administration include, but are not limitedto, ingestion and rectal routes and can include the use of, for example,pharmaceutically acceptable powders, pills or liquids for ingestion andsuppositories for rectal administration.

Naso-pharyngeal and pulmonary administration include are accomplished byinhalation, and include delivery routes such as intranasal,transbronchial and transalveolar routes. The invention includesformulations of carrier suitable for administration by inhalationincluding, but not limited to, liquid suspensions for forming aerosolsas well as powder forms for dry powder inhalation delivery systems.Devices suitable for administration by inhalation of carrierformulations include, but are not limited to, atomizers, vaporizers,nebulizers, and dry powder inhalation delivery devices.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

In some embodiments, the synthetic, biodegradable particles of thepresent invention provide ease of manufacturing, broad availability oftherapeutic agents, and increased treatment sites. In particularembodiments, surface-functionalized biodegradablepoly(lactide-co-glycolide) particles with a high density of surfacecarboxylate groups, synthesized using the surfactantpoly(ethylene-alt-maleic anhydride) provide a carrier that offersnumerous advantages over other carrier particles and/or surfaces.Experiments conducted during development of embodiments of the presentinvention demonstrated the conjugation of peptides (e.g., PLP₁₃₉₋₁₅₁peptide) to these particles. Such peptide-coupled particles have shownthat they are effective for the prevention of disease development andthe induction of immunological tolerance (e.g., in the SJL/JPLP₁₃₉₋₁₅₁/CFA-induced R-EAE murine model of multiple sclerosis).Peptide coupled carriers of the present invention provide numerousadvantages over other tolerance induction structures. In someembodiments, the particles are biodegradable, and therefore will notpersist for long times in the body. The time for complete degradationcan be controlled. In some embodiments, particles are functionalized tofacilitate internalization without cell activation (e.g.,phosphatidylserine loaded into PLG microspheres). In some embodiments,particles incorporate targeting ligands for a specific cell population.In some embodiments, anti-inflammatory cytokines such as IL-10 andTGF-β, are included on or within particles to limit activation of thecell type that is internalizing the particles and to facilitate theinduction of tolerance via energy and/or deletion and the activation ofregulatory T cells. The composition of the particles has been found toaffect the length of time the particles persist in the body andtolerance requires rapid particle uptake and clearance/degradation.Since ratios of over 50:50 lactide:glycolide slow the degradation rate,the particles of the invention have a lactide:glycolide ratio of about50:50 or below. In one embodiment the particles of the invention haveabout a 50:50 D,L-lactide:glycolide ratio.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the modifiedparticles are mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The modified particles can also be in micro-encapsulated form with oneor more excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose and starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the modified particles only,or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

The present invention encompasses pharmaceutically acceptable topicalformulations of the inventive modified particles. The term“pharmaceutically acceptable topical formulation”, as used herein, meansany formulation which is pharmaceutically acceptable for intradermaladministration of modified microparticles of the invention byapplication of the formulation to the epidermis. In certain embodimentsof the invention, the topical formulation comprises a carrier system.Pharmaceutically effective carriers include, but are not limited to,solvents (e.g., alcohols, poly alcohols, water), creams, lotions,ointments, oils, plasters, liposomes, powders, emulsions,microemulsions, and buffered solutions (e.g., hypotonic or bufferedsaline) or any other carrier known in the art for topicallyadministering pharmaceuticals. A more complete listing of art-knowncarriers is provided by reference texts that are standard in the art,for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and17th Edition, 1985, both published by Mack Publishing Company, Easton,Pa., the disclosures of which are incorporated herein by reference intheir entireties. In certain other embodiments, the topical formulationsof the invention may comprise excipients. Any pharmaceuticallyacceptable excipient known in the art may be used to prepare theinventive pharmaceutically acceptable topical formulations. Examples ofexcipients that can be included in the topical formulations of theinvention include, but are not limited to, preservatives, antioxidants,moisturizers, emollients, buffering agents, solubilizing agents, otherpenetration agents, skin protectants, surfactants, and propellants,and/or additional therapeutic agents used in combination to the modifiedparticles. Suitable preservatives include, but are not limited to,alcohols, quaternary amines, organic acids, parabens, and phenols.Suitable antioxidants include, but are not limited to, ascorbic acid andits esters, sodium bisulfite, butylated hydroxytoluene, butylatedhydroxyanisole, tocopherols, and chelating agents like EDTA and citricacid. Suitable moisturizers include, but are not limited to, glycerine,sorbitol, polyethylene glycols, urea, and propylene glycol. Suitablebuffering agents for use with the invention include, but are not limitedto, citric, hydrochloric, and lactic acid buffers. Suitable solubilizingagents include, but are not limited to, quaternary ammonium chlorides,cyclodextrins, benzyl benzoate, lecithin, and polysorbates. Suitableskin protectants that can be used in the topical formulations of theinvention include, but are not limited to, vitamin E oil, allantoin,dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the invention comprise at least the modified particlesof the invention and a penetration enhancing agent. The choice oftopical formulation will depend or several factors, including thecondition to be treated, the physicochemical characteristics of theinventive compound and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplaryembodiments, penetration agents for use with the invention include, butare not limited to, triglycerides (e.g., soybean oil), aloe compositions(e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyl laurate, glycerol monooleate, and propyleneglycol monooleate) and N-methylpyrrolidone.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred. Creams of theinvention may also contain a non-ionic surfactant, for example,polyoxy-40-stearate. In certain embodiments, the active component isadmixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. As discussed above, penetration enhancingagents can also be used to increase the flux of the compound across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix or gel.

The modified particles can be administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the modified particles. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics®, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

It will also be appreciated that the modified particles andpharmaceutical compositions of the present invention can be formulatedand employed in combination therapies, that is, the compounds andpharmaceutical compositions can be formulated with or administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, an inventive compoundmay be administered concurrently with another anti-inflammatory agent),or they may achieve different effects (e.g., control of any adverseeffects).

In certain embodiments, the pharmaceutical compositions containing themodified particles of the present invention further comprise one or moreadditional therapeutically active ingredients (e.g., anti-inflammatoryand/or palliative). For purposes of the invention, the term “Palliative”refers to treatment that is focused on the relief of symptoms of adisease and/or side effects of a therapeutic regimen, but is notcurative. For example, palliative treatment encompasses painkillers,antinausea medications and anti-sickness drugs.

The invention provides methods of regulating an immune response in anindividual, preferably a mammal, more preferably a human, comprisingadministering to the individual the modified particles described herein.Methods of immunoregulation provided by the invention include those thatsuppress and/or inhibit an innate immune response or an adaptive immuneresponse, including, but not limited to, an immune response stimulatedby immunostimulatory polypeptides or viral or bacterial components.

The modified particles are administered in an amount sufficient toregulate an immune response. As described herein, regulation of animmune response may be humoral and/or cellular, and is measured usingstandard techniques in the art and as described herein.

In some embodiments, compositions described herein are administeredalong with (e.g., concurrent with, prior to, or following) an implant(e.g., device) and/or transplant (e.g., tissue, cells, organ) tomediate, negate, regulate and/or reduce the immune response associatedtherewith.

In certain embodiments, the individual suffers from a disorderassociated with unwanted immune activation, such as allergic disease orcondition, allergy and asthma. An individual having an allergic diseaseor asthma is an individual with a recognizable symptom of an existingallergic disease or asthma. Tolerance can be induced in such anindividual, for example, by particles complexed with the specific foods(e.g. peanut proteins, etc.), injected substances (e.g. bee venomproteins, etc.), or inhaled substances (e.g. ragweed pollen proteins,pet dander proteins, etc.) which elicit the allergic reaction.

In certain embodiments, the individual suffers from a disorderassociated with unwanted immune activation, such as autoimmune diseaseand inflammatory disease. An individual having an autoimmune disease orinflammatory disease is an individual with a recognizable symptom of anexisting autoimmune disease or inflammatory disease. Tolerance can beinduced in such an individual, for example, by particles complexed withthe relevant autoantigens driving the particular autoimmune disease.

In certain embodiments, the individual suffers from a disorderassociated with enzyme replacement therapy. Tolerance can be induced insuch an individual, for example, by particles complexed with the enzymeswhich patients with genetic deficiencies fail to produce, to preventthem from making neutralizing antibody responses torecombinantly-produced enzymes administered to treat their particulardeficiency, e.g. tolerance to human Factor VIII in patients withhemophilia due to a genetic deficiency in the ability to make FactorVIII. Another example may include enzyme replacement in for conditionssuch as mucopolysaccharide storage disorder, gangliosidosis, alkalinehypophosphatasia, cholesterol ester storage disease, hyperuricemia,growth hormone deficiency, renal anemia or with lysomal storagedisorders including Fabry's disease, Gaucher's disease, Hurler'sdisease, Hunter's syndrome, Maroteaux-Lamy disease, Niemann-Pickdisease, Tay-Sachs disease, and Pompe disease.

In certain embodiments, the individual suffers from a robust, orotherwise adverse, immune response towards an agent administered for thetreatment of a disease that actually or potentially compromises patienthealth or treatment. Additionally, novel compounds provided by thisinvention may be provided to individuals who do not show an immuneresponse to an agent but may potentially do so in the future. In certainembodiments, the individual is receiving enzyme replacement therapy. Incertain embodiments, therapeutic agents include, but are not limited to,peptides or protein-based agents, DNA vaccines, siRNA, splice-siteswitching oligomers, and RNA-based nanoparticles. In some embodiments,the therapeutic agents include, but are not limited to, Advate,antihemophilic factor, Kogenate, Eloctate, recombinant factor VIII Fcfusion protein, Refacto, Novo VIIa, recombinant factor VII, eptacogalfa, Helixate, Monanine, Coagulation Factor IX, Wilate, Ceredase,Alglucerase, Cerezyme, Imiglucerase, Elelso, taliglucerase alfa,Fabrazyme, Agalsidase beta, Aldurazyme, -I-iduronidase, Myozyme,Acid-glucosidase, Elaprase, iduronate-2-sulfatase, Naglazymearylsufatase B, and N-acetylgalactosamin e-4-sulfatase. In someembodiments, the individual is administered therapeutic agentsadministered to treat diseases including, but not limited to,Hemophilia, Hemophilia A, Hemophilia B, von Willebrand disease,Gaucher's Disease, Fabry's Disease, Hurler's Disease, Pompe's Disease,Hunter's Disease, mucopolysaccharide storage disorder, gangliosidosis,alkaline hypophosphatasia, cholesterol ester storage disease,hyperuricemia, growth hormone deficiency, renal anemia andMaroteaux-Lary Disease.

In certain embodiments, the individual suffers from an orphan autoimmunecondition. Such conditions include, but are not limited to, idiopathicthrombocytopenic purpura, membranous nephropathy, bullous pemphigoid,pemphigus vulgaris, and Myasthenia Gravis.

In certain embodiments, the individual suffers from a disorderassociated with disease therapy. In the case of recombinant antibodies,tolerance is induced for example, to a humanized antibody being employedin a therapeutic context to prevent a patient from making neutralizingantibodies against the antibody therapeutic, e.g. tolerance to ahumanized immune subset depleting antibody or anti-cytokine antibodybeing used as a treatment for autoimmune disease.

Autoimmune diseases can be divided in two broad categories:organ-specific and systemic. Autoimmune diseases include, withoutlimitation, rheumatoid arthritis (RA), systemic lupus erythematosus(SLE), type I diabetes mellitus, type II diabetes mellitus, multiplesclerosis (MS), immune-mediated infertility such as premature ovarianfailure, scleroderma, Sjögren's disease, vitiligo, alopecia (baldness),polyglandular failure, Grave's disease, hypothyroidism, polymyositis,pemphigus vulgaris, pemphigus foliaceus, inflammatory bowel diseaseincluding Crohn's disease and ulcerative colitis, autoimmune hepatitisincluding that associated with hepatitis B virus (HBV) and hepatitis Cvirus (HCV), hypopituitarism, graft-versus-host disease (GvHD),myocarditis, Addison's disease, autoimmune skin diseases, uveitis,pernicious anemia, Celiac disease, hypoparathyroidism neuomyelitisoptica, membraneous nephropathy, bullous pemphigoid, pemphigus vulgaris,myasthenia gravis.

Autoimmune diseases may also include, without limitation, Hashimoto'sthyroiditis, Type I and Type II autoimmune polyglandular syndromes,paraneoplastic pemphigus, bullus pemphigoid, dermatitis herpetiformis,linear IgA disease, epidermolysis bullosa acquisita, erythema nodosa,pemphigoid gestationis, cicatricial pemphigoid, mixed essentialcryoglobulinemia, chronic bullous disease of childhood, hemolyticanemia, thrombocytopenic purpura, Goodpasture's syndrome, autoimmuneneutropenia, myasthenia gravis, Eaton-Lambert myasthenic syndrome,stiff-man syndrome, acute disseminated encephalomyelitis, Guillain-Barresyndrome, chronic inflammatory demyelinating polyradiculoneuropathy,multifocal motor neuropathy with conduction block, chronic neuropathywith monoclonal gammopathy, opsonoclonus-myoclonus syndrome, cerebellardegeneration, encephalomyelitis, retinopathy, primary biliary sclerosis,sclerosing cholangitis, gluten-sensitive enteropathy, ankylosingspondylitis, reactive arthritides, polymyositis/dermatomyositis, mixedconnective tissue disease, Bechet's syndrome, psoriasis, polyarteritisnodosa, allergic anguitis and granulomatosis (Churg-Strauss disease),polyangiitis overlap syndrome, hypersensitivity vasculitis, Wegener'sgranulomatosis, temporal arteritis, Takayasu's arteritis, Kawasaki'sdisease, isolated vasculitis of the central nervous system,thromboangiutis obliterans, sarcoidosis, glomerulonephritis, andcryopathies. These conditions are well known in the medical arts and aredescribed, for example, in Harrison's Principles of Internal Medicine,14th ed., Fauci A S et al., eds., New York: McGraw-Hill, 1998.

Animal models for the study of autoimmune disease are known in the art.For example, animal models which appear most similar to human autoimmunedisease include animal strains which spontaneously develop a highincidence of the particular disease. Examples of such models include,but are not limited to, the nonobese diabetic (NOD) mouse, whichdevelops a disease similar to type 1 diabetes, and lupus-like diseaseprone animals, such as New Zealand hybrid, MRL-Fas^(lpr) and BXSB mice.Animal models in which an autoimmune disease has been induced include,but are not limited to, experimental autoimmune encephalomyelitis (EAE),which is a model for multiple sclerosis, collagen-induced arthritis(CIA), which is a model for rheumatoid arthritis, Desmoglein 3transgenic T cell mouse, which can be used as an experimental model ofPemphigus Vulgaris and experimental autoimmune uveitis (EAU), which is amodel for uveitis. Animal models for autoimmune disease have also beencreated by genetic manipulation and include, for example, IL-2/IL-10knockout mice for inflammatory bowel disease, Fas or Fas ligand knockoutfor SLE, and IL-I receptor antagonist knockout for rheumatoid arthritis.

In certain embodiments, the individual suffers from a bacterial or viralinfection. An individual having a bacterial or viral infection is anindividual with a recognizable symptom of an existing bacterial or viralinfection.

A non-limiting list of viral infections treatable with the modifiedparticles of the current invention includes herpes virus infections,hepatitis virus infections, west nile virus infections, flavivrusinfections, influenza virus infections, rhinovirus infections,papillomavirus infections, paromyxovirus infections, parainfluenza virusinfections, and retrovirus infections. Preferred viruses are thoseviruses that infect the central nervous system of the subject. Mostpreferred viruses are those that cause hemorragic fever, encephalitis ormeningitis.

A non-limiting list of bacterial infections treatable with the modifiedparticles of the current invention include staphylococcus infections,streptococcus infections, mycobacterial infections, bacillus infections,Salmonella infections, Vibrio infections, spirochete infections, andNeisseria infections. Preferred are bacteria that infect the centralnervous system of the subject. Most preferred are bacteria that causeencephalitis or meningitis.

In some embodiments, the invention relates to uses of compositions ofthis invention prior to the onset of disease. In other embodiments, theinvention relates to uses of the compositions of this invention toinhibit ongoing disease. In some embodiments, the invention relates toameliorating disease in a subject. By ameliorating disease in a subjectis meant to include treating, preventing or suppressing the disease inthe subject.

In some embodiments, the invention relates to preventing the relapse ofdisease. For example, an unwanted immune response can occur at oneregion of a peptide (such as an antigenic determinant). Relapse of adisease associated with an unwanted immune response can occur by havingan immune response attack at a different region of the peptide. Sincethe immune modifying particles of the current invention are free fromattached peptides or antigenic moieties, the particles will be effectiveagainst multiple epitopes. T-cell responses in some immune responsedisorders, including MS and other ThI/17-mediated autoimmune diseases,can be dynamic and evolve during the course of relapsing-remittingand/or chronic-progressive disease. The dynamic nature of the T-cellrepertoire has implications for treatment of certain diseases, since thetarget may change as the disease progresses. Previously, pre-existingknowledge of the pattern of responses was necessary to predict theprogression of disease. The present invention provides compositions thatcan prevent the effect of dynamic changing disease, a function of“epitope spreading.” A known model for relapse is an immune reaction toproteolipid protein (PLP) as a model for multiple sclerosis (MS).Initial immune response can occur by a response to PLP139-15. Subsequentdisease onset can occur by a relapse immune response to PLP[pi]s-iβi.

Other embodiments of this invention relate to transplantation. Thisrefers to the transfer of a tissue sample or graft from a donorindividual to a recipient individual, and is frequently performed onhuman recipients who need the tissue in order to restore a physiologicalfunction provided by the tissue. Tissues that are transplanted include(but are not limited to) whole organs such as kidney, liver, heart,lung; organ components such as skin grafts and the cornea of the eye;and cell suspensions such as bone marrow cells and cultures of cellsselected and expanded from bone marrow or circulating blood, and wholeblood transfusions.

A serious potential complication of any transplantation ensues fromantigenic differences between the host recipient and the engraftedtissue. Depending on the nature and degree of the difference, there maybe a risk of an immunological assault of the graft by the host, or ofthe host by the graft, or both, may occur. The extent of the risk isdetermined by following the response pattern in a population ofsimilarly treated subjects with a similar phenotype, and correlating thevarious possible contributing factors according to well acceptedclinical procedures. The immunological assault may be the result of apre-existing immunological response (such as preformed antibody), or onethat is initiated about the time of transplantation (such as thegeneration of Th cells). Antibody, Th cells, or Tc cells may be involvedin any combination with each other and with various effector moleculesand cells. However, the antigens which are involved in the immuneresponse are generally not known, therefore posing difficulties indesigning antigen-specific therapies or inducing antigen-specifictolerance.

Certain embodiments of the invention relate to decreasing the risk ofhost versus graft disease, leading to rejection of the tissue graft bythe recipient. The treatment may be performed to prevent or reduce theeffect of a hyperacute, acute, or chronic rejection response. Treatmentis preferentially initiated sufficiently far in advance of thetransplant so that tolerance will be in place when the graft isinstalled; but where this is not possible, treatment can be initiatedsimultaneously with or following the transplant. Regardless of the timeof initiation, treatment will generally continue at regular intervalsfor at least the first month following transplant. Follow-up doses maynot be required if a sufficient accommodation of the graft occurs, butcan be resumed if there is any evidence of rejection or inflammation ofthe graft. Of course, the tolerization procedures of this invention maybe combined with other forms of immunosuppression to achieve an evenlower level of risk.

Certain embodiments of the invention relate to decreasing or otherwiseameliorating the inflammatory response induced as a response to surgery.In one embodiment of the invention, the immune-modifying particles areadministered before surgery. In a further embodiment of the invention,the immune-modifying particles are administered concurrently with orduring surgery. In yet a further embodiment of the invention, theimmune-modifying particles are administered after surgery.

The particles of the invention may also be used to treat abscesses orempyemas to decrease the inflammatory response produced in the subjectafter exposure to infectious agents such as bacteria or parasites. Inone embodiment of the invention, the immune-modifying particles areadministered in conjunction with anti-bacterial and/or anti-parasitictreatments known in the art.

The particles of the invention may also be used to decrease or otherwiseameliorate the inflammatory response induced as a response to physicaltrauma or injury including, but not limited to, a sports injury, awound, a spinal cord injury, a brain injury, and/or a soft tissueinjury. In one embodiment of the invention, the immune-modifyingparticles are administered after the subject experiences trauma orinjury.

The particles of the invention may also be used to decrease theinflammatory response associated with the development and/or growth ofcancer cells. Cancers that can be treated include, but are not limitedto, central nervous system cancer, basal cell carcinoma, cancerous braintumors, Burkitt's lymphoma, lymphoma, cervical cancer, ovarian cancer,testicular cancer, liver cancer, non-small cell and small cell lungcancers, melanoma, bladder cancer, breast cancer, colon and rectalcancers, endometrial cancer, kidney (renal cell) cancer, leukemia,Non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, melanoma, andthyroid cancer. In one embodiment, the subcutaneous injection of theparticles of the invention prevents the accumulation of inhibitoryneutrophils, thereby decreasing inflammation in the cancer patient.

The particles of the invention are also useful for the regeneration ofdamaged tissue. In one embodiment, administration of the particles to apatient increases the regeneration of damaged epithelial cells in thedigestive tract. In a further embodiment, the patient suffers fromcolitis, Crohn's disease, or inflammatory bowel disease. In anotherembodiment, administration of the particles of the invention to apatient increases remyelination of neurons. In a further embodiment, thepatient suffers from multiple sclerosis.

In some embodiments, compositions of the present invention (e.g., PLGcarrier coupled to antigenic molecule) find use with one or morescaffolds, matrices, and/or delivery systems (See, e.g., U.S. Pat. App.2009/0238879; U.S. Pat. Nos. 7,846,466; 7,427,602; 7,029,697; 6,890,556;6,797,738; 6,281,256; herein incorporated by reference in theirentireties). In some embodiments, particles (e.g., antigen-coupled PLGparticles) are associated with, adsorbed on, embedded within, conjugatedto, etc. a scaffold, matrix, and/or delivery system (e.g., for deliveryof chemical/biological material, cells, tissue, and/or an organ to asubject). In some embodiments, a scaffold, matrix, and/or deliverysystem (e.g., for delivery of chemical/biological material, cells,tissue, and/or an organ to a subject) comprises and/or is made frommaterials described herein (e.g., PLG conjugated to one or moreantigenic peptides).

In some embodiments, microporous scaffolds (e.g., for transplantingbiological material (e.g., cells, tissue, etc.) into a subject) areprovided. In some embodiments, microporous scaffolds are provided havingthereon agents (e.g., extracellular matrix proteins, exendin-4) andbiological material (e.g., pancreatic islet cells). In some embodiments,the scaffolds are used in the treatment of diseases (e.g., type 1diabetes), and related methods (e.g., diagnostic methods, researchmethods, drug screening). In some embodiments, scaffolds are providedwith the antigen-conjugated carriers described herein on and/or withinthe scaffold. In some embodiments, scaffolds are produced from antigenconjugated materials (e.g., antigen conjugated PLG).

In some embodiments, a scaffold and/or delivery system comprises one ormore layers and/or has one or more chemical and/or biologicalentities/agents (e.g., proteins, peptide-conjugated particles, smallmolecules, cells, tissue, etc.), see, e.g., U.S. Pat. App. 2009/0238879;herein incorporated by reference in its entirety. In some embodiments,antigen-coupled particles are co-administered with a scaffold deliverysystem to elicit induction of immunological tolerance to the scaffoldand the associated materials. In some embodiments, microporous scaffoldis administered to a subject with particles described herein on orwithin the scaffold. In some embodiments, antigen-coupled particlescoupled to a scaffold delivery system. In some embodiments, a scaffolddelivery system comprises antigen-coupled particles.

Various modification, recombination, and variation of the describedfeatures and embodiments will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughspecific embodiments have been described, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes andembodiments that are obvious to those skilled in the relevant fields areintended to be within the scope of the following claims. For example,U.S. Pat. Applications 2012/0076831, 2002/0045672, 2005/0090008,2006/0002978, and 2009/0238879 (each of which is herein incorporated byreference in their entirety) and U.S. Pat. Nos. 7,846,466; 7,427,602;7,029,697; 6,890,556; 6,797,738; and 6,281,256 (each of which is hereinincorporated by reference in their entirety) provide details,modifications, and variations that find use in various embodimentsdescribed herein.

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference in their entireties.

EXAMPLES

The following examples are provided to further illustrate the advantagesand features of the invention, but are not intended to limit the scopeof this disclosure.

Materials and Methods

Generation of Chimeric Mice

Six- to eight-week old B6.SJL-Ptprc^(a)Pep3^(b)/BoyJ (CD45.1) mice wereirradiated with one dose of 950 rads. Twelve hours later, mice werereconstituted with 10⁷ bone marrow cells from C57BL/6-7.2fms-EGFPdonors. Mice were given sulfamethoxazole (Sigma Aldrich) andtrimethoprim (Sigma Aldrich) in the drinking water for 10 days followingirradiation. Mice were infected with WNV six weeks after irradiation, asdescribed above. Chimerism was checked using flow cytometry and wasinvariably found to be 96-99% of donor origin as previously demonstrated(Getts et al., J Neurochem. 103: 1019, 2007).

Immunohistology

Mice were anesthetized and perfused with 50 mL sterile PBS. With theexception of the heart, which were processed into paraffin blocks (Gettset al., J. Neurochem 103:10919-1030, 2007), all organs were isolated andsnap frozen in Optimum Cutting Temperature Compound (OCT; Tissue-Tek,Tokyo, Japan). Eight-micron tissue sections were cut on a cryostatmicrotome, air-dried overnight and then stored at −80° C. untilrequired. Frozen sections were thawed and histology (standardhaematoxylin and eosin staining) or immunohistochemistry was performed(Getts et al., J. Exp Med 205:2319-2337, 2008). Antibodies againstMARCO, SIGN-R1 and SIGLEC-1 (R&D Systems, MN, USA), CD68 (Abcam, MA,USA) and Ki67 (Abcam), were used as indicated. Images were acquired onan Olympus BX-51 microscope using a DP-70 camera and DP manager 2.2.1software (Olympus, Tokyo, Japan).

Microscope and Image Acquisition

Images were acquired on an Olympus BX-51 microscope (Olympus, Japan),using a DP-70 camera and DP manager 2.2.1 software (Olympus).

Isolation of Leukocytes from the Brain and Liver

As previously described (Getts et al, J Exp Med. 29: 2319, 2007)leukocytes were obtained from the brains of PBS-perfused mice bydigesting brains for 60 minutes at 37° C. in PBS with deoxy-ribonuclease(0.005 g/ml; Sigma Aldrich) and collagenase IV (0.05 g/ml; SigmaAldrich). Digestion was stopped with 10% FCS, and the homogenate waspassed through a 70 μm nylon cell strainer (Becton Dickinson, NJ, USA).The pellet, obtained after 10 minutes centrifugation at 340×g, wasresuspended in 30% Percoll (Amersham, Norway) and layered over 80%Percoll. Leukocytes were collected from the 30%/80% interface aftercentrifugation at 1140×g for 25 minutes at room temperature. The sameprotocol is also used to derive leukocytes from the liver, with thetissue weighed before processing.

Isolation of Leukocytes from the Spleen, Blood and Bone Marrow

For flow cytometric analysis, the right femur was dissected out and bonemarrow cells flushed out using PBS loaded syringes. For bone marrowprecursor isolation, femurs and tibias from at least 4 mice wereutilized. The cellular suspension achieved after flushing was filteredthrough a 70 μm cell strainer and centrifuged for 5 mins at 340 g. Redblood cells in the resulting pellet were lysed in NH₄Cl-based red celllysis buffer (BD Pharm Lyse™; BD Pharmingen), before centrifugation for5 mins at 340×g. In the case of peripheral blood, blood was collectedvia cardiac puncture and immediately transferred into citrate buffer(mMol, Sigma Alrich). The resulting suspension was layered over 70%Percoll and centrifuged at 1140×g for 20 minutes at room temperaturewith the brake off. The interface was collected and the cells washedonce in PBS, centrifuged at 340×g. For the isolation of splenicleukocytes, spleens were passed through a 7070 μm cell strainer andcentrifuged for 5 mins at 340 g. Red blood cells in the resulting pelletwere lysed in NH₄Cl-based red cell lysis buffer (BD Pharm Lyse™; BDPharmingen), before centrifugation for 5 mins at 340×g.

Flow Cytometry

Cells collected (as described above) from the brain, liver, blood, andbone marrow were washed in PBS, and blocked with anti-CD16/CD32 antibody(Biolegend). Viable cells were counted using trypan blue exclusion,which routinely showed >95% cell viability.

Cell surface molecule expression was measured and cell sorts carried outon a FACS ARIA (Becton Dickinson), equipped with an Argon ion and HeNelaser. Viable populations were gated by forward and side scatter andidentified fluorescent populations determined by forward-gatingthereafter. Sorting was carried out using specific fluorescent andscatter parameters identifying the population of interest. Sortingstringencies was set to purity to achieve >98% purity for bone marrowpopulations.

Acquired FACS data files were analysed using the flow cytometry program,Flow Jo (FlowJo, Ashland, Oreg., USA). Quantification of cellpopulations of interest were calculated based on flow cytometrypercentages at analysis and absolute cell counts from each organ.

Adoptive Transfer

Experiments were conducted during development of embodiments of thepresent invention to investigate a second model of active disease termedadoptive transfer. Rather than immunizing the animals with the peptide,the lymphocytes from the spleen of mice with active disease weretransferred to a recipient, who would subsequently develop disease.Experiments were conducted during development of embodiments of thepresent invention to characterize the ability of the PLG nanoparticlesto deactivate the adoptively transferred activated effector cells. Micetreated with particles or splenocytes coupled with a control peptide hadan increase in clinical score beginning at day 4. Mice treated withPLG-PLP₁₃₉₋₁₅₁ particles at day 2 had a mean clinical score of 0 for allbut two time points through day 40, and the mean clinical score forthose other time points was 0.25.

Multiplex ELISA

Multiplexed plate ELISAs were performed according to the manufacturersinstructions (Quansys Biosciences, Logan, Utah, USA). Briefly, brain,spleen, and liver tissue were homogenized in PBS, clarified by a 1000×gspin, and stored at −20° C. until the assay was performed. Serum sampleswere also used. Thawed samples and standards were diluted in theprovided buffer, and 30 μl of each were plated in each well thatcontains 16 spots each containing a capture antibody for a particularsoluble protein. Plates were then incubated for 1 hour on an orbitalshaker at 120 r.p.m. Plates were washed 3 times, and 30 μl of detectionantibody was added to each well and incubated for another hour. Afterwashing 3 times, strepavidin-HRP was added and incubated for a further15 minutes. Plates were then washed 6 times, and substrate mix wasadded. Plates were immediately read on a CCD imager (Kodak, RochesterN.Y., USA). Plate images were analysed using Quansys Q-view software(Quansys Biosciences).

Induction and Evaluation of Experimental Autoimmune Encephalitis (EAE)

Mice were injected sub-cutaneously with emulsion containing 0.1 mg MOGPeptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:1); Auspep, Parkville,Victoria, Australia; >95% HPLC purified) and Complete Freund's adjuvantcontaining 2 mg/mL Mycobacterium tuberculosis (Sigma Aldrich). Two dayslater, mice were administered 500 μl Pertussis toxin (Sigma Aldrich)i.p. Mice were monitored for disease progression, and graded on thefollowing scale: 1, limp tail and/or weakness of 1 hind limb; 2,weakness in more than one limb, gait disturbance; 3, paralysis in 1limb; 4, paralysis in more than one limb, incontinence; 5, moribund.

Statistics

Graphs were made and computerized statistical analysis was performed inGraphPad Prism, and InStat, respectively (both programs from GraphPadsoftware, San Diego, Calif., USA). Depending on the data, an unpaired,two-tailed Student t-test, or one way ANOVA with a Tukey-Kramer posttest was performed, with P<0.05 considered to be significant.

For correlation analysis between parameters such as weight loss,infiltration, and virus titre, a non-linear regression (curve fit) wasused, with a second order polynomial (Y=A+B*X+C*X{circumflex over( )}2).

Example 1

Preparation of Negatively Charged Immune Modifying Particles (IMPs)

To a solution of Poly(ethylene-maleic anhydride) (PEMA) in D₂O (4 mL, 1%w/v) was added dropwise a solution of poly(lactide-co-glycolic acid)(PLG) in dichloromethane (DCM) (2 mL, 20% w/v). The mixture was allowedto sonicate on ice at 16 watts for 30 sec using the VC 30 UltrasonicProcessor. The resulting homogenized crude was then poured into asolution of D₂O (200 mL containing 0.5% w/v of PEMA). The homogenizedslurry was allowed to stir overnight at speed setting of 3.5 usingBellco Glass, Inc., Bellstir Multi-stir 9 magnetic stirrer (10 W for 10s, 16 W for 10 s, 16 W for 30 s).

Results

After three hours of stirring, particle size analyses were performedusing dynamic light scattering in disposable polystyrene cuvettes

-   -   a. 10 W, 10 s−Z-average=499.9 nm−PdI=0.23, Peak=634.5 nm    -   b. 16 W, 10 s−Z-average=528.9 nm−PdI=0.227, Peak=657.5 nm    -   c. 16 W, 30 s−Z-average=471.6 nm−PdI=0.228, Peak=580.5 nm    -   d. 16 W, 60 s−Z-average=491.1 nm−PdI=0.275, Peak=600.8 nm

After the reaction was complete, the resulting crude suspension was thenpurified.

Purification

Fresh D₂O and 10× sodium bicarbonate buffer were chilled overnight to 4°C. Using a 40 μm cell strainer, 36 mL of particle suspension werefiltered from each batch into an appropriately-labelled 50 mL centrifugetube containing 4 mL chilled 10× sodium bicarbonate buffer. Each beakerproduced approximately 6 such tubes. All tubes were centrifuged forabout 15 minutes at 7000 g at 4° C. and the supernatant was aspirated.Preparation of the suspension was repeated using the above-mentionedprocedure and much of the particle pellets were suspended as possible in1 mL chilled D₂O.

The resuspended particles were transferred into a fresh tube with 4 mLof chilled 10× sodium bicarbonate buffer. (Step 1)

Resuspension of the particle was repeated until the entire particlepellets haves been successfully resuspended. (Step 2)

The 6 centrifugal tubes were then combined into one centrifuge tube (50mL tube) and the tube was filled with the remaining volume to 40 mL ofchilled D₂O (Wash 1).

The tube was centrifuged for 20 minutes at 7000 g at 4° C. and thesupernatant was aspirated.

Step 1 and 2 and Wash 1 of the resulting particle were repeated eachtime at least two more times. Finally, the resulting particle pelletswere then subjected to a flash-freeze in liquid nitrogen and lyophilizedto dryness in the manifold to obtain negatively IMPs.

FIG. 1 shows characterization of surface-functionalizedpoly(lactide-co-glycolide) particles by dynamic light scatteringanalysis. Surface-functionalized poly(lactide-co-glycolide) particleswere analysed on a Malvern Zetasizer Nano ZS (Malvern Instruments,Westborough, Mass.) at a count rate of 2.5×10⁵ counts per second in 18.2MΩ water. The population of surface-functionalizedpoly(lactide-co-glycolide) particles had a Z-average diameter of 567 nm,a peak diameter of 670 nm and a polydispersity index of 0.209.

Table 4 shows the measurements for surface functionalized PLG-PEMAparticles. The data in the table is representative, as each batch isslightly different. The numbers in the table were based on combiningseveral batches of particles though. The measurements for the doubleemulsion particles are similar to those in Table 2.

TABLE 4 Measurements for the surface functionalized PLG-PEMA particlesZ-average size by Particle intensity (nm) ζ-potential (mV) PLG(Phosphorex) 624.3 −32.7 ± 4.71 PLG-PEMA 429.9 −67.4 ± 10.9

Example 2

Administration of Antigen-Coupled PLGA Beads Prevents RelapsingExperimental Autoimmune Encephalitis

PLG nanoparticles were investigated with the immunodominant proteolipidprotein PLP₁₃₉₋₁₅₁ epitope (PLG-PLP₁₃₉₋₁₅₁) to induce tolerance forprevention of Relapsing Experimental Autoimmune Encephalitis (R-EAE).The R-EAE mice were generated as described above.

The peptides administered to the animals were coupled to particles withthe mean diameter of 500 nm. Mice were treated with eitherPLP₁₃₉₋₁₅₁-PLGA (N=5), OVA₃₂₃₋₃₃₉-PLGA (N=5), or unconjugated PLGA (N=5)on day −7 relative to the time of immunization (day 0). Peak disease wastypically observed around day 12 to 14, and mice are scored for clinicaldisease. Particles without peptide, or modified with the control peptideOVA₃₂₃₋₃₃₉ did not prevent disease induction. However, PLGA particlesmodified with PLP₁₃₉₋₁₅₁ produced a clinical score of 0 (no disease) atall except low clinical scores of under 1 exhibited between days 20 and30 (FIG. 2). Previous studies with unmodified PLG or using polystyreneparticles did not produce this effective disease reduction, withpolystyrene bound particles commonly triggering anaphylaxis.

Furthermore, specific inactivation of myelin-specific CD4⁺ T cells wasdemonstrated by lack of delayed-type hypersensitivity (DTH) responses toboth immunizing PLP₁₃₉₋₁₅₁ epitope. Taken together, prophylactictreatment with PLG-PLP₁₃₉₋₁₅₁ on day-7 specifically prevented EAEdevelopment, and represents an improvement in the ability of particlesto prevent disease. The scores produced with the particles are as goodas, and perhaps better, than the scores produced with antigen-coupledsplenocytes.

The type of particle administered also has an effect on the developmentof EAE in the mouse model. Mice were treated with either OVA₃₂₃₋₃₃₉-PLS(N=5), OVA₃₂₃₋₃₃₉-PLGA_(PHOSPOREX) (N=5), OVA₃₂₃₋₃₃₉-PLGA_(PEMA) (N=5),PLP₁₃₉₋₁₅₁-PLA (N=5), PLP₁₃₉₋₁₅₁-PLGA_(PHOSPOREX) (N=5), orPLP₁₃₉₋₁₅₁-PLG_(PEMA) (N=5) on day −7 relative to the time ofimmunization (day 0). Peak disease was typically observed around day 12to 14, and mice are scored for clinical disease. Particles, of anycomposition that were modified with the control peptide OVA₃₂₃₋₃₃₉ didnot prevent disease induction. However, the PLP₁₃₉₋₁₅₁ coupled PLG beadswere more effective in down-regulating induction of R-EAE thanPLP₁₃₉₋₁₅₁ coupled commercial (Phosphorex) pLG or polystyrene (FIGS. 3Aand 3B).

Example 3

Intravenous Infusion of Antigen Coupled PLG Particles does not InduceAnaphylaxis-Induced Temperature Drop in OVA/Alum Pre-Sensitized Animals

Due to the presence of active disease, anaphylaxis to the antigens is aconcern, which could result in immediate mortality, and has beendescribed with polystyrene bound particles. Anaphylaxis is associatedwith a significant drop in body temperature. To test whether intravenousadministration of OVA-PLG induces an anaphylaxis-induced temperaturedrop in pre-sensitized animals, mice were immunized at day 0 with 10 μgOVA/Alum via intraperitoneal injection. On day 14, the mice were againimmunized with 10 μg OVA/Alum via intraperitoneal injection, and thentolerized with OVA-PLG administered intravenously on day 21. On day 28,the mice were then tolerized with either OVA-PLG particles or OVA viaintravenous administration.

As shown in FIG. 4 those mice treated with soluble OVA on day 28exhibited decrease in temperature compared with those animals treatedwith the OVA-PLG particle. No decrease in body temperature was observedwithin 1 hour of delivering the particles.

FIG. 5 shows that administration of PLP-PLG during remission does notresult in any anaphylaxis-associated mortality. EAE was induced in sixto eight week old female SJL/J mice by subcutaneous injection ofPLP₁₃₉₋₁₅₁ in CFA, and development of clinical disease was monitored andrecorded (FIG. 5B). On day 21 relative to disease induction, mice weregiven iv injections of soluble PLP₁₃₉₋₁₅₁ (clear squares), solubleOVA₃₂₃₋₃₃₉ (clear circles), or the same peptides coupled to PLGnanoparticles (solids). Temperature of animals was monitored andrecorded every 10 minutes for 1 hour following injection (FIG. 5A).

Example 4

Prophylactic Treatment with PLP-PLG Particles Induces Long-Term,Antigen-Specific Tolerance

Optimal dosing was determined by intravenous administration ofincreasing concentrations of PLP₁₃₉₋₁₅₁-PLG seven days prior to diseaseinduction, and monitored for development of clinical disease incomparison to SJL/J mice treated with OVA₃₂₃₋₃₃₉-PLG (FIG. 6A). Six toeight week old female SJL/J mice were injected iv with either PLP₁₃₉₋₁₅₁(square)- or OVA₃₂₃₋₃₃₉ (circle)-coupled PLG nanoparticles. EAE wasinduced by subcutaneous injection of PLP₁₃₉₋₁₅₁ in CFA 7 days (FIG. 6B),25 days (FIG. 6C), or 50 days (FIG. 6D) later. Animals from panel B werefollowed for clinical disease for 100 days. FIG. 6E shows that on day 8relative to disease induction, a delayed-type hypersensitivity (DTH)reaction was carried out in a subset of the mice shown in panel B.Selected representative animals from the PLP₁₃₉₋₁₅₁/CFA primed groups inpanel B (OVA₃₂₃₋₃₃₉-PLG and PLP₁₃₉₋₁₅₁-PLG) were ear-challenged with thepriming PLP₁₃₉₋₁₅₁ epitope and the OVA₃₂₃₋₃₃₉ control peptide. Earswelling as a measure of DTH was determined 24 h later and responsesprior to challenge were subtracted. FIG. 6F shows that six to eight-weekold female SJL/J mice were injected intravenously with PLP₁₇₈₋₁₉₁(triangle)-, OVA₃₂₃₋₃₃₉ (circle), or PLP₁₃₉₋₁₅₁ (square)-coupled PLGnanoparticles, or with uncoupled particles alone (outlined circle). EAEwas induced 7 days afterward by subcutaneous injection of PLP₁₇₈₋₁₉₁ inCFA, and disease was monitored at the time points shown.

Example 5

Treatment of Relapsing Experimental Autoimmune Encephalitis withAntigen-Coupled Particles

Experiments were conducted during development of embodiments of thepresent invention to investigate the ability of the PLG-PLP₁₃₉₋₁₅₁particles to treat disease rather than prevent disease, and to determinewhether the route of administration affected the development of disease.Mice were immunized at day 0 with PLP₁₃₉₋₁₅₁ and an adjuvant. Micenormally have maximal clinical scores at day 12-14. In this model, themice were treated at day 10 with the PLG-PLP₁₃₉₋₁₅₁ particles or withcontrol PLG-OVA₃₂₃₋₃₃₉ particles either via intravenous (iv)administration, intraperitoneal (ip) administration, subcutaneous (sc)administration, or orally. As shown in FIG. 7, prophylactic tolerance ismost efficient when the PLG-PLP₁₃₉₋₁₅₁ particles are administered eitherintravenously or intraperitoneally. Animals treated with PLP₁₃₉₋₁₅₁-PLGadministered intravenously did not develop disease and had mean clinicalscores of 0 at most time points. This is in contrast to animals treatedwith PLP₁₃₉₋₁₅₁ polystyrene particles, whereby >70% of animals whereobserved to die from anaphylaxis.

Example 6

Antigen-Coupled Particle Tolerance Inhibits Induction ofAntigen-Specific Th1 and Th17 Responses in Active Relapsing ExperimentalAutoimmune Encephalitis

To determine whether administration of antigen-coupled particles inhibitinduction of T-helper cells, either MOG₃₅₋₅₅-PLG or OVA₃₂₃₋₃₃₉-PLGparticles were administered intravenously to BALB/c mice at Day −7. OnDay 0, OVA₃₂₃₋₃₃₉-PLG particles and Complete Freund's Adjuvant (CFA)were administered subcutaneously to the mice. The animals werere-stimulated with either MOG₃₅₋₅₅-PLG or OVA₃₂₃₋₃₃₉-PLG particles onDay 10 and the draining lymph node cells were isolated. The CPM andlevels of IL-17, GM-CSF, IFN-γ, IL-10, and IL-4 were measured at Day 10.As shown in FIG. 8, the administration of OVA₃₂₃₋₃₃₉-PLG particlesinhibited the Th1 and Th17 responses in the treated animals.

Example 7

Tolerance is Induced by PLP-₁₃₉₋₁₅₁ Coupled PLGA Particles

An additional therapeutic tolerance strategy was performed by deliveringPLP₁₃₉₋₁₅₁-PLG or OVA₃₂₃₋₃₃₉ PLG to mice. Histological analysis showedthat the administration of the PLP₁₃₉₋₁₅₁-PLG particles inhibitscervical spinal cord inflammation and demyelination. Mice were treatedwith PLP-PLG or OVA₃₂₃₋₃₃₉-PLG and the tissue was retrieved at day 40.The cervical spinal cord was isolated and sectioned to investigate theimmune response within the CNS, which underlies the pathology of R-EAEand multiple sclerosis. FIG. 9 shows a reduction in immune cellinfiltration within the spinal cord of animals treated withPLP₁₃₉₋₁₅₁-PLG that and was more similar to native tissue than to tissuefrom OVA₃₂₃₋₃₃₉-PLG treated animals. OVA₃₂₃₋₃₃₉-PLG treated animals hadpositive staining for CD45, CD4, and CD11b; whereas, PLP₁₃₉₋₁₅₁-PLGtreated animals had minimal staining for these factors.

Administration of PLP₁₃₉₋₁₅₁-PLG particles also inhibits blood brainbarrier (BBB) disruption and macrophage activation in the spinal cord oftreated mice. Animals were treated with Complete Freund's Adjuvant(CFA), OVA₃₂₃₋₃₃₉ PLG particles, or PLP₁₃₉₋₁₅₁-PLG particles. Theclinical scores and percent incidence of EAE were determined (FIG. 10B)and the spinal cords observed via in vivo imaging (FIGS. 10A and 11).Angiosense measures vascular leak in the CNS and prosense reportsactivated macrophages (cathepsin activation cleaves the reporterrevealing the fluorescent signal). The bar graphs give the numericalnumbers to the signal strength shown in the brain and SC scans.

Tolerance can also be induced by particles in which the antigen has beenencapsulated. FIG. 12 shows that the administration of PLG particles inwhich PLP₁₃₉₋₁₅₁ has been encapsulated inhibits the induction of R-EAEin mice. The ability to encapuslate autoantigens allows for the used ofcomplex mixtures of proteins or even organ homogenates to achieve moreantigen coverage and thus more effectively deal with epitope spreading.

Example 8

Tolerance Induced by PLP-₁₃₉₋₁₅₁ Coupled PLGA Particles is PartiallyDependent on the Expansion/Activation of Regulatory T-Cells

SJL/J mice were treated with an anti-CD25 antibody, a common marker forregulatory T cells (Tregs) on Day −9, and then on Day −7 were treatedwith either OVA₃₂₃₋₃₃₉ PLG particles and anti-CD25 antibody, OVA₃₂₃₋₃₃₉PLG particles and a control IgG antibody, PLP₁₃₉₋₁₅₁-PLG particles andan anti-CD25 antibody, or PLP₁₃₉₋₁₅₁-PLG particles and a control IgGantibody. As shown in FIG. 13, animals treated with the PLP₁₃₉₋₁₅₁-PLGparticles and the anti-CD25 antibody demonstrated, at times, a greatermean clinical score than those animals treated with PLP₁₃₉₋₁₅₁-PLGparticles and a control IgG antibody. This confirms that Tregs, or atleast T cells expressing CD25, play a role in the initiation oftolerance.

Example 9

Therapeutic Tolerance is Induced by PLP₁₃₉₋₁₅₁-PLG Particles in Activeand Adoptive EAE

Therapeutic tolerance induced by PLP₁₃₉₋₁₅₁-PLG particles was comparedin active and adoptive EAE. Adoptive EAE was induced in six toeight-week old female SJL/J mice by adoptive transfer of 2.5×10⁶PLP₁₃₉₋₁₅₁-activated blasts. Mice were injected iv with PLP₁₃₉₋₁₅₁(squares) or OVA₃₂₃₋₃₃₉ (circles) peptide coupled to 500 nm PLGnanoparticles 2 days (FIG. 14A), 14 days (FIG. 14C), 18 days (FIG. 14E),or 21 days (FIG. 14F) following disease induction. Clinical diseasescores were compared to those following treatment with antigen-coupledsplenocytes (FIG. 14A). Brain and spinal cord were collected fromPLP₁₃₉₋₁₅₁- or OVA₃₂₃₋₃₃₉-tolerized mice for histological analysis onday 42. Sections from mice from panel A were stained for PLP protein andCD45 (FIG. 14B). Spinal cord sections from mice from panel C werestained with Luxol Fast Blue (FIG. 14D). Areas of demyelination andcellular infiltration are indicated by arrows. The results show thattolerance is induced by PLP₁₃₉₋₁₅₁-PLG particles in mice with adoptiveEAE.

FIG. 15 shows graphs depicting the mean clinical scores of mice withactive EAE and adoptive EAE after treatment with either SP or PLGparticles conjugated to OVA₃₂₃₋₃₃₉ or PLP₁₃₉₋₁₅₁. Mice were injected ivwith PLP₁₃₉₋₁₅₁-SP, PLP₁₃₉₋₁₅₁-PLG, or OVA₃₂₃₋₃₃₉-SP, or OVA₃₂₃₋₃₃₉-PLGpeptide coupled to 500 nm nanoparticles 10 days (FIG. 15A) or 2 days(FIG. 15B) following disease induction and the mean clinical score wasdetermined. In both cases, administration of PLP₁₃₉₋₁₅₁-PLG particlesreduced disease, indicative of tolerance induction.

The infiltration of central nervous system immune cells is alsodrastically reduced in PLP-PLG tolerized mice. SJL/J mice were injectedi.v. with 500 nm PLG nanoparticles coupled with PLP₁₃₉₋₁₅₁ (squares) orOVA₃₂₃₋₃₃₉ (circles) 2 days following EAE induction by adoptivetransfer. At the peak of disease (day 14) brains and spinal cords wereremoved and the number of lymphocytes (FIG. 16B), APCs (FIG. 16C),microglia (FIG. 16D), peripheral dendritic cells (FIG. 16E), myeloiddendritic cells (FIG. 16F) and macrophages (FIG. 16G) were enumerated byflow cytometry. The gating strategy for these populations is depicted in(FIG. 16A). CNS cell preparations were stimulated with PMA and ionomycinfor 5 h prior to intracellular staining for IL-17A and IFN-γ (FIG. 16H).

Example 10

Treatment with an Anti-PD-1 Monoclonal Antibody Abrogates ToleranceInduction with PLG Nanoparticles Encapsulating PLP₁₃₉₋₁₅₁ in AdoptiveTransfer EAE

To test the effect of treatment with an anti-PD-1 antibody on PLP₁₃₉₋₁₅₁induced tolerance in mice with adoptive EAE, on Day 0, mice received3×10⁶ PLP₁₃₉₋₁₅₁ activated T-cell blasts via intravenous administration.On Day 2, they received PLP₁₃₉₋₁₅₁ or OVA₃₂₃₋₃₃₉ encapsulated in PLGparticles via intravenous administration with either PBS or an anti-PD-1antibody. On days 4, 6, 8, 10, and 12 all animals received either 250 μganti-PD-1 antibody or PBS.

As shown in FIG. 17, administration of the PLP₁₃₉₋₁₅₁ peptideencapsulated in a PLG particle induces tolerance when the particle isadministered with PBS. However, administration of the anti-PD-1 antibodydecreases this tolerance.

Example 11

Treatment with an Agonistic Anti-CD40 Monoclonal Antibody AbrogatesTolerance Induction with PLG Nanoparticles Encapsulating PLP₁₃₉₋₁₅₁ inAdoptive Transfer EAE in an IL-12 Dependent Manner

To test the effect of treatment with an agonistic anti-CD40 antibody onPLP₁₃₉₋₁₅₁ induced tolerance in mice with adoptive EAE, on Day 0, micereceived 3×10⁶ PLP₁₃₉₋₁₅₁ activated T-cell blasts via intravenousadministration. On Day 2, the mice received PLP₁₃₉₋₁₅₁ or OVA₃₂₃₋₃₃₉encapsulated in PLG particles via intravenous administration. On day 3,the animals received a control IgG2a antibody, an anti-CD40 antibody, oran anti-CD40 antibody and an anti-Il-12 antibody.

As shown in FIG. 18, administration of the PLP₁₃₉₋₁₅₁ peptideencapsulated in a PLG particle induces tolerance when the particle isadministered with PBS. Administration of the agonistic anti-CD40antibody decreases this tolerance, but this decrease in tolerance isreversed by the addition of an anti-IL-12 antibody.

Example 12

OVA Encapsulated in PLG Particles Prophylactically Inhibits AllergicAirway Inflammation and In Vivo OVA-Specific Th2 Responses

To test the prophylactic effect of OVA encapsulated in PLG particles onairway inflammation, mice were treated intravenously with OVA-PLG at day−7. On day 0, the mice received intraperitoneal injections of OVA/Alumat a dose of 10 μg/mouse. On day 7, the mice were again treatedintravenously with OVA-PLG and received another 10 μg/mouse ip injectionof OVA/Alum on day 14. Between days 28 and 30, the mice were treatedthree times with aerosolized OVA.

As shown in FIG. 19, the prophylactic administration of OVA-PLGdecreased the secretion of IL-4, IL-5, IL-13 and IL-10, and reduced thelevels of serum OVA IgE and eosinophils in the lung.

OVA Encapsulated in PLG particles prophylactically inhibits OVA-specificin vitro recall responses from mediastinal lymph nodes. As shown in FIG.20A, the lymph node proliferation observed after restimulation with 25μg OVA is decreased in those animals treated with OVA-PLG. Moreovertreatment with OVA-PLG decreases the release of cytokines afterrestimulation with OVA. FIG. 20B shows that levels of IL-4, IL-5, IL-13,and IL-10 are decreased in mice treated with OVA-PLG.

Example 13

OVA Encapsulated in PLG Particles Therapeutically Inhibits AllergicAirway Inflammation and In Vivo OVA-Specific Th2 Responses

To test the therapeutic effect of OVA encapsulated in PLG particles onairway inflammation, mice were treated intraperitoneally with OVA/Alumat a dose of 10 μg/mouse on day 0 and day 14. The mice wereintravenously administered with OVA-PLG on days 28, and 42. Between days56-58, the mice were treated three times with aerosolized OVA.

As shown in FIG. 21, the therapeutic administration of OVA-PLG decreasedthe secretion of IL-4, IL-5, IL-13 and IL-10, and reduced the levels ofserum OVA IgE and eosinophils in the lung.

FIG. 22 shows OVA Encapsulated in PLG particles therapeuticallydown-regulates OVA-Specific Th2 Cytokines in the BAL Fluid Better thanOVA-coupled PLG particles. The animals were treated as described aboveexcept that on days 28 and 42, the mice were treated with either OVAencapsulated in PLG particles, or OVA coupled to PLG particles.Surprisingly, the encapsulated OVA inhibited the secretion of Th2cytokines more than the OVA peptide coupled to the surface of the PLGparticle.

Example 14

Tolerance Induced by Chromogranis A p31 Peptide-PLG Particles InhibitsType 1 Diabetes

Type 1 diabetes was induced in BDC2.5 mice by isolating spleen,axillary, brachial, inguinal, and pancreatic lymph node cells from miceat 3 weeks. The isolated cells were cultured and activated in vitro byincubating 2×10⁶ cells/mL with 0.5 μM p31 peptide for 96 hours. 5×10⁶cells were transferred via intravenous administration to NOD.SCID mice(6-8 weeks) at Time 0. The mice were tolerized via intravenousadministration with p31 or MOG₃₅₋₅₅ peptide coupled to SP or PLG 2 hoursto 3 days later.

FIGS. 23A and 23B show the blood glucose levels in the animals aftertreatment. Administration of the p31 peptide coupled PLG resulted inlower blood glucose levels compared to those seen after administrationwith the MOG₃₅₋₅₅ peptide coupled particles. FIG. 23C shows that thepercent of IFNγ secreting cells observed in the animals was also reducedin the p31-PLG treated mice compared with the MOG₃₅₋₅₅ peptide-PLGtreated mice.

p31-PLG induced tolerance requires Tregs. Type 1 diabetes was induced inmice as described above, and 2 hours after the activated cells weretransferred to the NOD.SCID mice, the mice were tolerized with eitherp31-PLG or MOG₃₅₋₅₅ PLG particles. As shown in FIG. 24, depletion ofTregs abrogates the tolerance induced by administration of p31-PLGparticles.

Example 15

Tolerance Induced by Insulin-Coupled PLG Particles Inhibits theDevelopment of Spontaneous Type 1 Diabetes in NOD Mice

NOD mice were treated with either BSA (N=22) or insulin (N=23) coupledPLG particles via intravenous administration at 6, 8, and 10 weeks ofage. The mice were then assayed for the development of diabetes whichwas defined as blood glucose >250 mg/dL. As shown in FIG. 25,administration of the insulin coupled PLG particles significantlyincreased the percentage of mice that did not develop diabetes over 300days (69.6% compared to 22.7%; p=0.0027).

Example 16

Engraftment Kinetics

Female CD45.2 mice were tolerized with either OVA-PLG or the controlpeptide Dby-PLG (the major H-Y antigen expressed by Male C57BL/6 mice)on day −7. On day −1, the mice were irradiated with 200 rads and werethen transplanted with 1×10⁶, 5×10⁶, or 1×10⁷ bone marrow cells frommale CD45.1 mice on day 0. The recipient mice were then tolerized witheither OVA-PLG, Dby-SP, or Dby-PLG on day 1 and the blood harvested forFACS analysis of chimerism. FIG. 26 shows the percent of CD45.1 donorcells observed in the recipient mice.

FIG. 27 shows the percent of donor CD45.1 cells in the recipient miceafter tolerization with either OVA-PLG, Dby-SP, or Dby-PLG on day 1. Onepositive control mouse did not demonstrate significant engraftment(˜10%). All negative control mice did not engraft donor cells. OneDby-SP mouse did not demonstrate significant engraftment (˜10%). TwoOVA-PLG mice engrafted donor cells (˜10%): one completely rejected byWeek 16. One Dby-PLG mouse started to reject at Week 12 and was at 10%by Week 16. The Dby-PLG group ranged from 10%-56% engraftment by Week16. The OVA-PLG mice demonstrated: 1) Spontaneous engraftment, 2)Sequence homology between OVA323 and Dby, or 3) tolerogenic propertiesof particles. Dby-PLG allows for more engraftment than Dby-SP andOVA-PLG.

FIG. 28 shows that the timing tolerance has an effect on the percent ofCD45.1 cells in the recipient mouse. Positive Controls show lessengraftment (˜4%) than expected (˜10%). One Negative control mouse had5% engraftment Out of all 3 OVA-PLG groups, one mouse in the Day −7, Day+1 group showed engraftment (12%). Tolerance on day 1 is more clinicallyrelevant than tolerance on day −7.

Example 17

Coumarin-6 PLGA Particles are not Detectable 24 Hours afterAdministration

Mice were treated with coumarin-6 PLGA particles that were eithercoupled to an antigen or antigen-free. As shown in FIG. 29, theparticles were detectable at 3 hours post-administration, but not at 24hours post-administration. Naïve uninjected mouse (top row) as comparedto i.v. fluorescent PLGA/PEMA microparticle injected mouse spleen (leftcolumn), liver (middle column) and lung (left column) sections at3-hours post injection (middle row) and 24-hours (bottom row)post-injection, counterstained with DAPI.

Example 18

Nanoparticles are Associated with Macrophages In Vivo

Analysis of the liver 6 hours and 15 hours post-administration showsthat PLGA particles co-localized with F4/80+ cells in the liver (FIG.30).

The marginal zone macrophages predominantly uptake TAMRA-labeledPLP₁₃₉₋₁₅₁-coupled particles 24 hours after intravenous infusion. Asshown in FIG. 31, the highest percentage of PLP₁₃₉₋₁₅₁+ cells aremarginal zone macrophages.

Example 19

Inhibition of R-EAE in SJL/J Mice Using Surface-FunctionalizedPoly(Lactide-Co-Glycolide) Particles Containing Soluble PLP₁₃₉₋₁₅₁within their Cores.

Groups of SJL/J mice were injected IV with 2.5 mg 500 nm-700 nmsurface-functionalized poly(lactide-co-glycolide) particles with solublePLP₁₃₉₋₁₅₁ peptide within their cores on Day −7 and Day −1 beforerelative to priming with PLP139-151/CFA on Day 0. Control mice wereprimed on Day 0 but did not receive particle treatment on Day −7 or Day−1. Mice were observed for clinical signs of R-EAE for an additional 20days.

The results depicted in FIG. 32 depict the daily mean clinical scoreagainst the number of days PLP₁₃₉₋₁₅₁/CFA priming.PLP₁₃₉₋₁₅₁/CFA-induced R-EAE is inhibited in SJL/J mice by the inductionof immunological tolerance using surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble PLP₁₃₉₋₁₅₁within their cores.

Example 20

Inhibition of Allergic Airway Inflammation by Surface-FunctionalizedPoly(Lactide-Co-Glycolide) Particles Containing Soluble Ovalbumin

Allergic airway inflammation (AIA) was induced in mice. Groups of Balb/cmice were injected intravenously with 2.5 mg 500 nm-700 nmsurface-functionalized poly(lactide-co-glycolide) particles with solubleovalbumin or soluble bovine serum albumin (control) within their coreson Day −7 and Day +7 before priming with ovalbumin/alum on Days 0 and+14. Mice were challenged on Days +28-30 with aerosolized ovalbumin.Mice were then sacrificed and bronchoalveolar lavage fluid obtained. Theserum levels of ovalbumin specific IgE were measured also.

Eosinophil counts within the bronchoalveolar lavage fluid indicate theseverity of AAI—higher counts indicated worse disease. Serum levels ofIgE indicate the severity of AAI—higher levels indicated worse disease.

FIG. 33 shows that mice treated with encapsulated OVA-PLG showed thegreatest reduction in eosinophil accumulation. FIG. 34 shows that micetreated with encapsulated OVA-PLG showed the greatest reduction in serumIgE levels compared to untreated or control treated animals.

Ovalbumin/alum-induced allergic airway inflammation in Balb/c mice wasinhibited by the induction of immunological tolerance usingsurface-functionalized poly(lactide-co-glycolide) particles containingsoluble ovalbumin within their cores.

Example 21

Synthesis of Surface-Functionalized Poly(Lactide-Co-Glycolide) ParticlesEncapsulating Antigen

The present Example details the formulation and partial characterizationof biodegradable poly(lactide-co-glycolide) particles that have beensurface-functionalized with a high density of carboxylate groups andcontain soluble antigen within their cores that are surrounded by ashell of poly(lactide-co-glycolide) for tolerance induction inautoimmune disease and for the treatment of allergies.

The high density of carboxylate groups was achieved by the use ofpoly(ethylene-alt-maleic anhydride (PEMA)), a polymer with carboxylategroups incorporated into its backbone, as the surfactant for theemulsification process.

As described above, biodegradable poly(lactide-co-glycolide) particlescontaining soluble PLP139-151 within their cores andsurface-functionalized with a high density of carboxylate groups areeffective for the induction of immunological tolerance in the SJL/JPLP139-151/CFA-induced R-EAE murine model of multiple sclerosis.Furthermore, biodegradable poly(lactide-co-glycolide) particlescontaining soluble ovalbumin within their cores andsurface-functionalized with a high density of carboxylate groups areeffective for the induction of immunological tolerance in the Balb/covalbumin/alum-induced AAI murine model of allergic asthma.

Poly(lactide-co-glycolide) particles containing soluble ovalbumin orbovine serum albumin within their cores and surface-functionalized witha high density of carboxylate groups were synthesized using a doubleemulsion-solvent evaporation method as follows:

1. 150 μL of 200 mg/mL ovalbumin or bovine serum albumin inendotoxin-free water was added dropwise to 2 mL of 20% w/vpoly(lactide-co-glycolide) in dichloromethane in a 20 mL scintillationvial.

2. The resultant mixture was placed on ice and sonicated for 30 secondsat 10 watts using a probe sonicator.

3. 10 mL of 1% w/v poly(ethylene-alt-maleic anhydride) in water wasadded.

4. The resultant mixture was placed on ice and sonicated for 30 secondsat 16 watts using a probe sonicator.

5. The resultant emulsion was poured into 200 mL 0.5% w/vpoly(ethylene-alt-maleic anhydride) in a 600 mL beaker and stirredovernight to allow for particle hardening.

6. The hardened particles were then purified by centrifugation andwashed 3 times with bicarbonate buffer pH 9.6.

7. The purified particles were resuspended in 4% w/v sucrose and 3% w/vD-mannitol in water, flash-frozen in liquid nitrogen and lyophilized todryness.

FIG. 35 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble PLP₁₃₉₋₁₅₁within their cores by dynamic light scattering analysis.Surface-functionalized poly(lactide-co-glycolide) particles wereanalyzed on a Malvern Zetasizer Nano ZS (Malvern Instruments,Westborough, Mass.) at a count rate of 1.792×105 counts per second in18.2 MΩ water. The population of surface-functionalizedpoly(lactide-co-glycolide) particles had a Z-average diameter of 584 nm,a peak diameter of 679 nm and a polydispersity index of 0.162. Theseresults are representative of 6 batches of syntheses, following theprotocol written above.

FIG. 36 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble PLP139-151within their cores by ζ-potential measurement. Surface functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 6.67×104 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had a peakζ-potential of −48.9 mV and a deviation of 5.14 mV. These results arerepresentative of 6 batches of syntheses, following the protocol writtenabove.

FIG. 37 shows the characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble ovalbumin withintheir cores by dynamic light scattering analysis. Surface-functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 1.822×105 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had aZ-average diameter of 569.7 nm, a peak diameter of 700.3 nm and apolydispersity index of 0.230. These results are representative of 3batches of syntheses, following the protocol written above.

FIG. 38 shows Characterization of surface-functionalizedpoly(lactide-co-glycolide) particles containing soluble ovalbumin withintheir cores by ζ-potential measurement. Surface functionalizedpoly(lactide-co-glycolide) particles were analyzed on a MalvernZetasizer Nano ZS (Malvern Instruments, Westborough, Mass.) at a countrate of 2.67×104 counts per second in 18.2 MΩ water. The population ofsurface-functionalized poly(lactide-co-glycolide) particles had a peakζ-potential of −52.2 mV and a deviation of 5.38 mV. These results arerepresentative of 3 batches of syntheses, following the protocol writtenabove.

Example 22

Surface-Functionalized Liposomes Containing Soluble PLP139-1.51 withintheir Cores Induce Immunological Tolerance in the Murine R-EAE Model ofMultiple Sclerosis

The present inventors have also discovered that biodegradable liposomaldelivery vehicles that have been surface-functionalized with a highdensity of negatively-charged groups and contain soluble antigen withintheir cores induce immunological tolerance in the R-EAE murine model ofmultiple sclerosis.

The liposomes used in this study were composed of the following lipidsat the following molar ratios—30:30:40phosphatidylcholine:phosphatidylglycerol:cholesterol. Groups of SJL/Jmice were injected IV with 200 nm surface-functionalized liposomes (10μmol total lipid per mouse) with soluble PLP₁₃₉₋₁₅₁ peptide within theircores on Day −7 relative to priming with PLP₁₃₉₋₁₅₁/CFA on Day 0.Control mice were primed on Day 0 and received 500 nm-700 nmsurface-functionalized liposomes (10 μmol total lipid per mouse) withsoluble OVA₃₂₃₋₃₃₉ peptide within their cores on Day −7. Mice wereobserved for clinical signs of R-EAE for an additional 17 days.

The results depict the daily mean clinical score against the number ofdays PLP₁₃₉₋₁₅₁/CFA priming. As shown in FIG. 39, the animals treatedwith the surface-functionalized liposomes with soluble PLP₁₃₉₋₁₅₁peptide within their cores had a lower clinical score than those animalstreated with the surface-functionalized liposomes containing solubleOVA₃₂₃₋₃₃₉ peptide.

The results of this study demonstrate that biodegradable liposomescontaining soluble PLP₁₃₉₋₁₅₁ within their cores andsurface-functionalized with high density of negatively-charged groupsare effective for the induction of immunological tolerance in the SJL/JPLP₁₃₉₋₁₅₁/CFA-induced R-EAE murine model of multiple sclerosis.

The tolerance induced by antigen-coupled or antigen-encapsulatedparticles is antigen-specific, dose dependent and long-lasting (>150days). Tolerance is best induced by intravenous administration of acoupled particle that is between 500 nm and 1 μm in diameter with a zetapotential <−5-mV. The induction of tolerance is dependent on the uptakeof the particles by the MARCO scavenger receptor with sees polyanionicsurfaces (e.g. carboxylated PS/PLG particles). The tolerance is inducedand maintained by a combination of anergy (partially reversed byanti-PD-1 and agonistic anti-CD40 antibodies) and iTregs (partiallyreversed by anti-CD25 antibodies). The particles of the inventionaccumulate predominantly in the liver and splenic marginal zonemacrophages (CD11b^(hu) CD11c^(lo) MARCO⁺ Sign-R1⁺ Siglec-1⁻).

There are numerous advantages of using antigen-coupled particles for thetreatment of autoimmune diseases compared with using antigen-pulsed orantigen-directed immature tolerogenic dendritic cells or engineeringantigen-specific Tregs. The rapidity and simplicity of tolerogenpreparation and induction using a GMP manufacturable, off-the-shelfuniversal tolerogenic carrier; there is no need to isolate and expandimmature dendritic cells or Tregs ex vivo; there is no need to beconcerned with immature dendritic cells being activated upon ex vivomanipulation and becoming stimulatory rather than tolerogenic or ofTregs converting to Th1/17 after transfer; since the hose immaturemarginal zone APCs process and represent the antigen in a tolerogenicmanner, host APCs can select the relevant immunodominant self epitopesfrom PLG particles encapsulating intact auto-antigens or tissue extracts(e.g. OVA encapsulated PLG particles prevent OVA/Alum-induced AAD); andthe protocol is antigen-specific with no bystander suppression, is safe,highly efficient, and can induce unresponsiveness in both effector Tcells (Th1, Th2, Th17, and CD8) and naïve T cells involved with epitopespreading.

Synthetic, biodegradable particles and liposomes could lead to ease ofmanufacturing, broad availability of therapeutic agents, and increasethe number of potential treatment sites. To this end, we havespecifically engineered surface-functionalized biodegradablepoly(lactideco-glycolide) particles with a high density of surfacecarboxylate groups, using the surfactant poly(ethylene-alt-maleicanhydride).

We have also developed surface-functionalized liposomes using a 30:30:40ratio of phosphatidylcholine:phosphatidylglycerol:cholesterol.

We have further engineered these particles to contain soluble ovalbuminwithin their cores so as to circumvent chemical contamination and purityissues surrounding surface-conjugation of peptide or protein. Thesesurface-functionalized poly(lactide-co-glycolide) particles containingsoluble ovalbumin within their cores are effective for the prevention ofdisease development and hence the induction of immunological tolerancein the Balb/c ovalbumin/alum-induced AAI murine model of allergicasthma. Peptide or protein conjugated to carboxylate-functionalizedpoly(lactide-co-glycolide) particles using EDC are attached in anindiscriminate fashion, resulting in antigen aggregates andparticle-antigen-particle aggregates that are difficult to characterizeand purify into homogeneous populations.

We have produced a homogeneous population of surface functionalizedpoly(lactide co-glycolide) particles containing soluble ovalbumin withintheir cores that do not require surface conjugation of antigen.

We have further demonstrated that biodegradable liposomes containingsoluble PLP₁₃₉₋₁₅₁ within their cores and surface-functionalized withhigh density of negatively-charged groups are effective for theinduction of immunological tolerance in the SJL/J PLP₁₃₉₋₁₅₁/CFA-inducedR-EAE murine model of multiple sclerosis.

The liposomes and poly(lactide-co-glycolide) particles of the presentinvention offer numerous advantages. The advantages include:

-   -   1) Biodegradable particles will not persist for long times in        the body, and the time for complete degradation can be        controlled.    -   2) Particles and liposomes can be functionalized to facilitate        internalization without cell activation. Toward this goal, we        have loaded phosphatidylserine into PLG microspheres.    -   3) Particles and liposomes can also be designed to incorporate        targeting ligands for a specific cell population.    -   4) Anti-inflammatory cytokines such as IL-10 and TGF-β, can also        be included to limit activation of the cell type that is        internalizing the particles and to facilitate the induction of        tolerance via anergy and/or deletion and the activation of        regulatory T cells.

This combinatorial function of the particle or liposome can targettolerance induction from multiple perspectives, thus designer particlesare a significant advance relative to the polystyrene particles.Potential clinical applications of this tolerance inducing technologyinclude:

-   -   (1) T cell- and antibody-mediated autoimmune diseases (such as        multiple sclerosis, type 1 diabetes, rheumatoid arthritis,        systemic lupus, etc.)—tolerance would be induced with particles        complexed with the relevant autoantigens driving the particular        autoimmune disease    -   (2) food and lung allergies, skin allergies, and        asthma—tolerance would be induced with particles complexed with        the specific foods (e.g. peanut proteins, etc.), injected (bee        venom proteins, etc.), or inhaled substances (e.g., ragweed        pollen proteins, pet dander proteins, etc.) which elicit the        allergic reaction    -   (3) transplant rejection—tolerance would be induced to the        transplant antigens on donor organs or cells prior to organ        transplant to prevent rejection by the recipient    -   (4) enzyme replacement therapy—tolerance would be induced to        enzymes which patients with genetic deficiencies fail to        produce, to prevent them from making neutralizing antibody        responses to recombinantly-produced enzymes administered to        treat their particular deficiency.

Example 23

The Particles Most Effective at Inducing Tolerance are NegativelyCharged and an Average Diameter of 500 nm

The key particle parameters for inducing tolerance are the size andcharge of the composition. As shown in FIGS. 40A and B, the charge ofthe particle affects the efficacy of tolerance induction. A comparisonof EAE mice treated with OVA conjugated particles with a −25 mv or −60mv charge found that compositions comprising particles with a charge of−60 mv induce tolerance more effectively than those with a −25 mVcharge. Mice were treated with TIMP (tolerogenic immune modifyingparticles) having a charge of either −60 mv or −25 mv. Mice were treatedwith either OVA₃₂₃₋₃₃₉-TIMP_(−60mv), OVA₃₂₃₋₃₃₉-PLGA_(−25mv),PLP₁₃₉₋₁₅₁-TIMP_(−60mv), or PLP₁₃₉₋₁₅₁-PLGA_(−25mv) (all antigens areencapsulated) and scored for clinical disease. Panel (A) shows the meanclinical score and Panel (B) shows the mean cumulative score of the EAEanimals.

The negative charge on the particle affects the ability of the particleto interaction with the MARCO scavenger receptor. FIG. 41 shows that thecharge of the immune-modifying particle is important for targeting theimmune modifying particle to the antigen presenting cell. Wild type orMARCO−/+ animals were treated with either PS-IMP or vehicle. The resultsindicate that particles with a reduced negative charge have a lowerefficacy because there is less interaction with the scavenger receptorssuch as MARCO that have positively charged collagen-like domains.

In addition to charge, the size and composition of the biodegradableTIMPs affects the induction of tolerance. As shown in FIG. 42A, the mosteffective particles for inducing tolerance in the EAE model are thosewith a mean diameter of about 500 nm. Mice were treated with either 500nm OVA₃₂₃₋₃₃₉-PSB, 100 nm PLP₁₃₉₋₁₅₁-PSB, 500 nm PLP₁₃₉₋₁₅₁-PSB, 1.75 μmPLP₁₃₉₋₁₅₁-PSB, or 4.5 μm PLP₁₃₉₋₁₅₁-PSB and scored for clinicaldisease. PLGA carriers have slow release kinetics for over 1 month andchanging the polymer ratio can impact release of the particles.Tolerance requires rapid particle uptake and clearance/degradation.Since ratios of over 50:50 lactide:glycolide slow the degradation rate,the particles of the invention in one embodiment are 50:50lactide:glycolide. FIG. 42B shows that the particles are rapidlydestroyed.

In addition to the charge and average mean diameter of the TIMPs,antigens which are encapsulated within the particle are superior toparticles coupled to antigen in the allergy model. In the allergy model,coupled nanoparticles have a propensity to cause anaphylaxis and are notan effective therapy. Conversely, as shown in FIG. 43, TIMPs with acharge of −60 mv are therapeutically effective in the murine allergymodel. Animals were exposed to OVA as an allergen, and were then treatedwith either a sham-PLG or TIMP particle, a PLG or TIMP particle withOVA, or no treatment. Panel (A) shows that OVA-PLG particles fail toreduce the TH2 response in allergy. Panel (B) shows thatTIMP_(PEMA-60mv) inhibit this TH2 response. Panel (C) shows thatTIMP_(PEMA-60mv) inhibit recall responses.

Example 24

Single-Emulsion Synthesis of Surface-FunctionalizedPoly(Lactide-Co-Glycolide) Particles Encapsulating Antigen

Polypeptide antigens can be incorporated into poly(lactide-co-glycolide)particles using a double-emulsion process (See, Example 21), however,the present inventors have found that when incorporating morehydrophobic polypeptides, such as gliaden, it is better to incorporatethe antigen into the particle with a single-emulsion process usingsolvents.

Poly(lactide-co-glycolide) with carboxylate end groups, a 50:50D,L-lactide:glycolide ratio, and inherent viscosity of 0.18 dl/g inhexafluoro-2-propanol was used to create particles containing gliaden.Poly(lactide-co-glycolide) particles containing gliaden within theircores and surface-functionalized with a high density of carboxylategroups were synthesized using a single emulsion-solvent evaporationmethod as follows:

-   -   1. Five milligrams of gliaden and 200 mg PLG was dissolved in 50        μL of trifluoroacetic acid (TFA) and 700 μL dimethylsulphoxide        and 1250 μL dichloromethane (DCM).    -   2. The resultant mixture was added drop-wise to 4 mL 1% w/v        aqueous PEMA and sonicated for 30 seconds at 100% amplitude.    -   3. The resultant emulsion was poured into 200 mL of 0.5% w/v        aqueous PEMA under stirring for 12 hours to allow the DCM to        completely evaporate.    -   4. The particles were then washed three times in 0.1M sodium        carbonate-sodium bicarbonate buffer pH 9.6. Alternatively, ddH₂O        can be used to wash the particles.    -   5. The purified particles were resuspended in 4% w/v sucrose and        3% w/v D-mannitol in water, gradually frozen to −80° C. and        lyophilized to dryness.

While specific embodiments of the invention have been described andillustrated, such embodiments should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims.

All patents, applications and other references cited herein areincorporated by reference in their entireties.

What is claimed is:
 1. A composition comprising surface functionalizedbiodegradable particles comprising one or more encapsulated antigens orantigenic epitopes thereof, wherein the particles have a negative zetapotential of about −100 mV to about −30 mV, and wherein the antigen isassociated with type-1 diabetes (T1D).
 2. The composition of claim 1,wherein the particles are poly(lactic acid) (PLA), poly(glycolic acid)(PGA), or poly(lactic co-glycolic acid) (PLGA) particles.
 3. Thecomposition of claim 1, wherein the antigens are selected from the groupconsisting pancreatic beta-cell antigen, insulin, proinsulin,islet-specific glucose-6-phosphatase catalytic subunit-related protein(IGRP), glutamic acid decarboxylase (GAD), zinc transporter 8 isoform a,and protein tyrosine phosphatase receptor type N.
 4. The composition ofclaim 3, wherein the insulin antigen or antigenic epitopes are selectedfrom the group consisting of SEQ ID: 1798, SEQ ID: 1987, SEQ ID: 4981,SEQ ID: 1971, SEQ ID: 2065, SEQ ID: 2240, SEQ ID: 2266, and SEQ ID:1839.
 5. The composition of claim 1, wherein the negative zeta potentialof the particles is achieved by surface functionalization.
 6. Thecomposition of claim 5, where the surface functionalization comprisescarboxylation.
 7. The composition of claim 1, wherein the particles arepoly(lactic co-glycolic acid) (PLGA) particles.
 8. The composition ofclaim 7, wherein the PLGA comprises a copolymer ratio of about 50:50 ofpolylactic acid:polyglycolic acid.
 9. The composition of claim 1,wherein the particles have a zeta potential between −30 mV and −80 mV.10. The composition of claim 9, wherein the particles have a zetapotential between −30 mV and −60 mV.
 11. The composition of claim 1,wherein the particles have a diameter between 200 nm and 2000 nm. 12.The composition of claim 11, wherein the particles have a diameterbetween 400 nm and 800 nm.
 13. The composition of claim 1 furthercomprising a pharmaceutically acceptable carrier or excipient.
 14. Thecomposition of claim 1 which is lyophilized.
 15. A compositioncomprising surface functionalized biodegradablepoly(lactide-co-glycolide) (PLG) particles comprising one or moreencapsulated antigens or antigenic epitopes thereof associated withtype-1 diabetes (T1D), wherein the PLG particles have a diameter ofabout 200 nm to about 2000 nm and a zeta potential of about −30 my toabout −80 my.
 16. A method of inducing antigen-specific tolerance in asubject having type-1 diabetes (T1D) comprising administering to thesubject an effective amount of the composition of claim
 1. 17. Themethod of claim 16, wherein the composition is administeredintravenously.
 18. A method of inducing antigen-specific tolerance in asubject having type-1 diabetes (T1D) comprising administering to thesubject an effective amount of the composition of claim
 15. 19. Thecomposition of claim 3, wherein the GAD antigens or antigenic epitopesare selected from the group consisting of SEQ ID: 1832, SEQ ID: 1841,SEQ ID: 1871, SEQ ID: 1875, SEQ ID: 1994, SEQ ID: 2012, SEQ ID: 2114,SEQ ID: 2249, SEQ ID: 2070 and SEQ ID:
 4982. 20. The composition ofclaim 3, wherein the IGRP antigen or antigenic epitope is set out in SEQID:
 1925. 21. The composition of claim 3, wherein the zinc transporter 8isoform a antigen or antigenic epitope is set out in SEQ ID:
 2421. 22.The composition of claim 3, wherein the protein tyrosine phosphatase,receptor type, N precursor antigen or antigenic epitope is set out inSEQ ID NO: 2028.